Methods and compositions for the treatment and diagnosis of shipping fever

ABSTRACT

Novel compositions are disclosed for use in the treatment or diagnosis of bovine pasteurellosis, commonly referred to as Shipping Fever. Cell-free Pasteurella haemolytica supernatants are employed to provide individual antigen compositions, identified through reaction with sera from naturally-infected or convalescent cattle. In particular, at least seven individual P. haemolytica antigen groups were recognized in cell-free culture supernatants. Purified P. haemolytica supernatant, formulated in a suitable pharmaceutical vaccine composition is shown to elicit a specific immune response, in both cows and rabbits, directed against the individual immunoreactive P. haemolytica polypeptides identified. Also disclosed are novel recombinant cells, plasmids and bacteriophage which include transcriptionally active P. haemolytica antigen genes. Recombinant clones are similarly selected to be reactive with naturally-infected antisera. Examples, and further disclosure, are also provided which demonstrate the utility of a presently disclosed antibody and antigen compositions in immuno-detection of both antigens and antibodies in various biological samples.

The present application is a divisional of U.S. Ser. No. 07/899,100filed Jun. 15, 1992 (now U.S. Pat. No. 5,336,491), which was acontinuation of U.S. Ser. No. 07/540,261 filed Jun. 18, 1990 (nowabandoned), which was a divisional of U.S. Ser. No. 07/085,430 filedAug. 13, 1987 (now U.S. Pat. No. 4,957,739), which was a continuation ofU.S. Ser. No. 06/935,806 filed Nov. 28, 1986 (now abandoned).

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is directed to methods and compositions forprophylaxis, treatment and diagnosis of pneumonic pasteurellosis incattle. More particularly, the present invention relates to theidentification and isolation of Pasterurella haemolytica antigens, usingboth recombinant DNA and non-recombinant technology, and the use of suchantigens in the formulation of antigen and antibody-containingcompositions for the treatment and diagnosis of pasteurellosis.

2. Description of the Related Art

Pneumonic pasteurellosis, commonly referred to as Shipping Fever, is amajor cause of economic loss in feedlot cattle. While there is evidenceto suggest that several etiologic agents, for example, combinations ofstress, respiratory viruses, and various bacteria may participate inthis disease, Pasterurella haemolytica, serotype A1, appears to be themajor cause of the severe fibrinous pneumonia which can be seen.

The pathogenesis of the disease is poorly understood. Overgrowth of thelung with P. haemolytica with resultant bronchial pneumonia is thoughtto be at least partly caused by a preceding viral infection. Studieshave suggested that parainfluenza-3 virus can impair pulmonary clearanceof P. haemolytica. Moreover, infectious bovine rhinotracheitis virus hasbeen shown to predispose pulmonary infection with P. haemolytica. Inexperimental bacterial infections in mice which have been pre-infectedwith virus, it has been shown that protection can be afforded by priorinoculation with either the viral or bacterial agent.

However, attempts to protect cattle by immunization with respiratoryviral vaccines and Pasteurella bacterins have generally provedunsuccessful. It has been proposed that antigenic challenge with deadbacteria, as is the case with bacterin immunization, is insufficient dueto the nature of the P. haemolytica infection--live P. haemolyticaapparently produce a cytotoxin having specificity for ruminantleukocytes. Thus, it is posited that following infection with P.haemolytica, the infected cow's immune system is suppressed to theextent that effective immunosurveillance is compromised and theinfective organism can not effectively be challenged. The failure ofPasteurella bacterins to provide an effective immunization has beenpartly ascribed to the absence of sufficiently antigenic amounts of thisleukotoxin in the bacterin preparation. The cytotoxin is thus believedto contribute to the pathogenesis of pneumonic pasteurellosis byimpairing primary lung defense and subsequent immune response, or byinduction of inflammation as a consequence of leukocyte lysis.

The physicochemical nature of the leukotoxin is only poorly understood.As noted, this toxin exerts no toxic effects on non-ruminant leukocytes.However, the toxic effects of the toxin on ruminant leukocytes is dosedependant--at lower doses, generally only subtle alterations in variousmetabolic processes are noted, whereas higher concentrations can resultin loss of membrane integrity and cell death. Apparent speciesspecificity of the leukotoxic effects of living P. haemolytica, andcell-free P. haemolytica supernatants, supports the hypothesis that theleukotoxin itself is involved in determining the species specificity ofthe Pasteurella-induced pneumonia. Moreover, experimental evidence fromstudies of the interactions of P. haemolytica and its culturesupernatant with ruminant alveolar macrophages, peripheral bloodmonocytes, neutrophils, and lymphocytes suggests that P. haemolyticaleukotoxin is important for successful colonization and growth of P.haemolytica in pulmonary tissues. Thus, cytotoxic effects of theleukotoxin for leukocytes in pulmonary tissues probably contribute tothe pathogenesis of the disease.

In contrast with bacterin immunization, immunization protocols employinglive P. haemolytica, and various protein extracts of P. haemolytica,have been shown to protect cattle against experimental challengeexposure to the bacterium. However, most of these studies involvedexperimental challenge exposure to live P. haemolytica organisms, ineither mice, where the organism induces a septicemia rather than arespiratory syndrome, or cattle, where the organism is aritificallyintroduced into the cattle's lungs. As such, neither of these testsystems represent a natural disease state, and are thus not believed toentirely correspond to natural pasteurellosis.

In 1985, Confer and Lessley investigated a series of saline proteincapsular extracts of P. haemolytica and identified a number of antigengroups through immuno-reaction with immune sera obtained from cows whichhad been immunized with live P. haemolytica organisms (Vet. Immunol. andImmunopath., vol. 10, pp. 265 and 279). Antibody response toimmunization with various of these capsular extracts was found tocorrelate with resistance to an experimental challenge of P. haemolyticaorganisms. However, as noted, these studies involved the use of capsular(i.e.--cell membrane) proteins which were then immunoidentified usingexperimentally induced antisera rather than antisera frompasteurellosis-infected cattle. Moreover, it is believed that the use ofcapsular proteins, rather than secreted proteins, and the use ofexperimentally induced antisera, rather than antisera from diseasedcattle, represent inherent drawbacks to such an approach to theidentification of antigens useful in the treatment of the disease.

Thus, attempts to develop a pasteurellosis vaccine to date have centeredon identifying the leukotoxin, or identifying antigens from proteinextraction of the P. haemolytica cell itself, rather than identifyingantigenic elements present in cell-free supernatants. However, thepresent invention, rather than focusing primarily on the leukotoxin,embodies the realization that cell-free supernatants contain numerousantigens--antigens which are necessarily absent or only minimallyrepresented in P. haemolytica bacterins--which should serve to induce amore effective immunization, or serve to complement, and therebyimprove, bacteria preparations. Moreover, the present invention embodiesthe further realization that effective P. haemolytica antigens should beidentified using antisera obtained from naturally-infected, active orconvalescent, cattle. The ultimate goal, therefore, is to achieve anantigenic composition which comprises a mixture or admixture ofindividual, relatively purified, P. haemolytica antigens whichcorrespond, at least in terms of antigenic determinants, to antigensidentified by antibodies present in naturally-infected antisera.

The present invention is thus directed in general to improved methodsfor identifying useful P. haemolytica antigens, one of which utilizesantisera from naturally-infected cattle to select antigens fromcell-free P. haemolytica culture supernatants, and the other employingrecombinant DNA technology to provide novel recombinant cells which areselected based on their ability to produce individual P. haemolyticaantigens as identified by the antisera.

SUMMARY OF THE INVENTION

Accordingly, the present invention in its most general scope is directedto the identification and isolation of antigenic P. haemolyticapolypeptides which may be employed, alone or in combination with eachother, in the formulation of compositions for the treatment and/orprevention of pasteurellosis. The identification and isolation ofantigenic polypeptides is achieved in two distinct fashions--byisolation of antisera-reactive antigenic proteins from cell-free P.haemolytica supernatants or through the use of recombinant DNAtechnology to construct recombinant cells which express individual P.haemolytica antigens. However, both approaches are related in that bothemploy antisera, from active or convalescent pasteurellosis-infectedcattle to identify antigens for immunogen formulation.

The first approach involves the identification of antigenic P.haemolytica polypeptides present in a cell-free P. haemolytica culturesupernatant. The use of such culture supernatants in the identificationof the antigens is believed to be particularly important to thesuccessful practice of the present invention in that culturesupernatants are enriched in those proteins which are released by theorganism ("extracellular" proteins) as opposed to those proteins whichare retained within the organism, or expressed only on its surface("intracellular" proteins). Thus, culture supernatant proteins arebelieved by the present inventors to correspond to those proteins whichare released from the bacteria during active P. haemolytica infectionsand are more likely to include, for example, leukotoxin componentsthought to be involved in the breakdown of immunosurveillance ininfected cattle, or other components not present in bacterins.

The supernatant approach to antigen isolation involves first culturingP. haemolytica bacteria to produce a culture supernatant which includesindividual P. haemolytica polypeptides. After removing the P.haemolytica cells from the culture supernatant, the resultant cell-freesupernatant is either employed directly or subjected to one of variousmolecular weight fractionation techniques known in the art, tofractionate the released P. haemolytica polypeptides according to theirmolecular weight. In one embodiment, SDS polyacrylamide gelelectrophoresis is employed to separate supernatant proteins accordingto their molecular weights for further identification, characterizationand purification.

For preparative separations, preparative gel electrophoresis isrecommended in that it has been determined that gel electrophoresisprovides the best separating capability for separating the antigensidentified in accordance with the present invention. However, otherseparating techniques may be employed, for example, gel exclusionchromatography, density gradient centrifugation, ion-exchange resins orhigh pressure liquid chromatography. Under specified gel exclusionchromatography of supernatant proteins reveals a highly immunoreactiveprotein complex in the exclusion volume. While this complex may beemployed directly in the formation of compositions, it may be alsoemployed as an initial step in the further purification supernatantantigens.

The next step includes identifying antigenic polypeptides byascertaining which of the extracellular (i.e.--supernatant) peptides arerecognized by antisera obtained from cattle which have manifesteddiscrete symptoms of the disease (e.g.--sniffling and wheezing,respiration distress, cough, fever, nasal discharge). Polypeptides whichare shown to react with sera from infected cattle have been found not toreact with "non-responder" control sera (i.e.--sera from non-infectedcattle). This finding ensures that such peptides are, in fact,Pasteurella antigens which are being specifically recognized bypasteurellosis-induced antibodies.

The specific technique employed by the present inventors to identify theP. haemolytica antigens is immunoblotting. Immunoblotting is a techniquewhich involves protein molecular weight fractionation, typically bypolyacrylamide gel electrophoresis, transferring the fractionatedproteins onto a nitrocellulose sheet, or other suitable adsorptionmatrix, and subjecting the sheet to a solution which includes theantisera under conditions which will allow for the formation of specificimunocomplexes between the Pasteurella-directed immunoglobulins presentin the antisera and antigens which have been adsorbed onto the sheet.The immunoreactive polypeptides may then be visualized through the useof a label, for example, in the form of a radioactive or enzymatic labelwhich has been attached to immunoglobulin molecules present in the sera,or to second antibody molecules which are specific for the sera. Bycomparing the gel migration distance of reactive peptides versus knownstandards, the molecular weight of the reactive peptides is ascertained.

Although the present invention is disclosed in terms of the immunoblottechnique, it will be recognized by those of skill in the art thatnumerous other techniques for identifying Pasteurella antigens may besuccessfully employed. Culture supernatant proteins may be fractionatedaccording to their molecular weight by any suitable technique. Forexample, column fractionation or density gradient centrifugation, asnoted above, may be employed wherein antigen identification is achievedby reacting column or gradient fractions with the immune sera, forexample, through use of ELISA or radioimmunoassay techniques.

After identification of the antigenic peptides, these peptides are thenselected for isolation where desired.

Isolation from polyacrylamide gels is achieved by excising the gelregions identified as containing the appropriate proteins, and elutingthe proteins from the gel, preferably by electroelution. Alternatively,where column or gradient fractionation is employed, the proteinfractions which exhibit the immunoreactivity are selected andindividually pooled.

Although preferred, there is no general requirement that the antigens beprovided in their most purified, gel-isolated, state. The antigens maybe provided either directly in the form of a purified culturesupernatant (i.e.--purified to remove small molecular weight anddialyzable contaminants, salts, etc.), or through further supernatantpurification schemes directed at partial purification by removingnon-antigenic proteins, while substantially retaining the antigens. Gelexclusion chromatography is one such technique which provides arelatively purified antigenic compositions, found to include most if notall of the dominant antigenic supernatant species.

An alternative approach which may be employed in the identification andisolation of supernatant antigens involves preparing an immunoaffinitychromatography substrate using immune sera from pasteurellosis-infectedcattle, and using the antibody-substrate to selectively purify theantigenic peptides. More particularly, immune sera fromPasteurella-infected cows is first attached to a substrate such asCNBr-Sepharose. The antisera-bound Sepharose is then poured into acolumn and washed with a suitable wash buffer. An aqueous mixture whichincludes the supernatant antigens is then passed over the column underconditions which allow for immunocomplex formation between the antigensin the mixture and the Sepharose-bound antibodies. After the column iswashed extensively to remove non-specifically bound material, thespecifically-bound antigens are then eluted from the column. ThisPasteurella-specific antigen mixture may then be employed directly orsize fractionated to further purify the individual antigens which may beidentified in accordance with the present disclosure.

By practicing one of the. foregoing cell-free supernatant-directedmethods, one may obtain a composition which includes one or moresubstantially purified P. haemolytica antigens which are secreted by theP. haemolytica cell, isolatable from a cell-free P. haemolytica culturesupernatant and have binding affinity for immune sera obtained from apasteurellosis infected cow. Such compositions may be identified asincluding antigens which are characterized according to apparentmolecular weight ranges which they exhibit upon SDS polyacrylamide gelelectrophoresis and immunoblot analysis.

Molecular weight assignments are approximated by correlating the SDS gelmigration of antigens to the migration of proteins of known molecularweights. Thus, differences in techniques for measuring migrationdistances will result in differences in apparent molecular weights.These differences are naturally accentuated by the fact that SDSpolyacrylamide gel electrophoresis is an inherently less accurate meansof determining the molecular weight of larger proteins. Moreover, someantigens have been found to exhibit broader banding patterns upon SDSgel electrophoresis, perhaps due to varying degrees of proteinmodifications, for example, glycosylation.

In particular, compositions are characterized as including at least oneof seven antigen groups, referred to as supernatant antigens I-VI,wherein the antigens exhibit molecular weight ranges as follows:

    ______________________________________                   Apparent                   Molecular Weight                               Reference    Antigen Group  Range       Weight    ______________________________________    I                     98-140K  105K    II                    86-110K  90K    III                  76-85K    76K    IV                   73-82K    73K    V                    63-71K    65K    VI                   42.5-45K  43K    VIIa                           35K                         29-35K    32K    c                              29K    ______________________________________

The "reference weight" above refers to the weight which represents theinventors best estimate of a specific molecular weight. As such, theparticular antigen groups may at times be referred to, for convenience,in terms of either the reference weight or the antigen groupdesignation. Such references should not be interpreted to limit thescope of the present invention to any such specific reference molecularweight and is meant to include the range as a whole.

It will also be appreciated that antigen group VII includes antigens ofthree separate molecular weight ranges. It has been found that allinfective P. haemolytica strains studied to date exhibit at least one,but generally not more than one, of these group VII antigens. Thus, itis hypothesized that these antigens are related, perhaps differing interms of glycosylation, amino acid sequence or other modifications.Hence, based on this hypothesis, these antigens have been groupedtogether as a single antigen group VII.

The individual reactive antigen groups, in the form of proteins, proteinfractions or mixtures, may thus be used to provide a composition whichis suitable for use as either a vaccine, or as an inoculum to generateantisera for use in, for example, providing passive immunity tohigh-risk cattle. Moreover, antibody compositions, derived from suchantisera or generated through hybridoma technology, may be used todiagnose pasteurellosis by using them to detect Pasteurella antigens inbiological fluids in the cow being tested.

As referred to above, a second general overall approach employed by thepresent inventors to identify P. haemolytica antigens involves the useof recombinant DNA technology. In this embodiment, recombinant cells areprovided which have been genetically engineered to individually expressindividual P. haemolytica antigens. Typically, such recombinant cellsinclude E. coli cells which have been transformed with an appropriaterecombinant cloning vector. The individual P. haemolytica antigens arethus coded for by P. haemolytica DNA fragments which have been ligatedto the cloning vector. The recombinant cell thereby expresses theantigen by expressing the protein coding sequences contained within thevector-ligated DNA fragments.

In particular, recombinant cells made in accordance with the presentinvention have been genetically engineered by a process which includesthe steps of fragmenting P. haemolytica DNA; ligating the fragmented DNAto a cloning vehicle, suitable for transforming a selected cell type, toproduce a recombined cloning vehicle; transforming the cell type withthe recombined cloning vehicle to produce transformed recombinant cells;selecting a clonal colony of the transformed recombinant cells whichproduces an individual P. haemolytica antigen; and culturing theselected clonal colony to provide the recombinant cell.

Fragmentation of the P. haemolytica DNA is employed in order to provideDNA fragments of a site range compatible with the particular cloningvector employed. For example, for most plasmids which are currently usedin the art, a size range of 1-15 kilobases is preferred. With fragmentsizes greater than 15 kilobases, recombinant plasmids are apparentlydestabilized. Bacteriophage vectors may accommodate much larger fragmentsizes, up to somewhat greater then 20 kilobase insertions, but generallyrequire a lower size limit of at least about 5 kilobases. The size rangelimitations for phage is generally dictated by their ability to besuccessfully "packaged" by phage coat proteins. Cosmids, a third generaltype of vector which may be employed, are composed of both phage andplasmid genetic elements and will generally accept size ranges of from 5to about 40 kilobases.

In one embodiment, the P. haemolytica DNA is randomly fragmented throughthe use of partial restriction enzyme digestions. In that suchdigestions are "partial", relatively large DNA fragments may be obtainedwhich contain full complements of genes. DNA fragments so-produced are"random" in that under "partial" restriction digestion conditions, notevery enzyme recognition site is recognized and cleaved. The fact that aselected restriction enzyme recognition site may be present within, forexample, a particular desired coding sequence does not limit theusefulness of "partial" enzyme digestion as a method for fragmenting theDNA because at least a proportion of the population of the DNA fragmentswill provide a full, uncleaved sequence of the particular gene. Thus,virtually any restriction enzyme may be employed for the generation ofP. haemolytica DNA fragments in accordance with the present invention.

However, it will be appreciated that there is no general requirementthat fragments be generated which contain entire coding sequences of aparticular P. haemolytica antigen gene. All that is required is toobtain fragments which are sufficiently long to code for polypeptidesequences which are recognized by pasteurellosis-derived antisera. Inthat they are "recognized" by the immune sera, such polypeptides will atleast contain the antigenic determinants ("epitopes") necessary torender such fragments antigenic and hence, such protein fragments may besuccessfully employed in vaccines or inoculums. For the purposes of thepresent invention, polypeptides which are derivations of full proteinsequences, but which polypeptides nevertheless include antigenicdeterminants and are therefore functional in providing an immuneresponse, are considered to be antigenic functional equivalents of thefull protein sequences and are thus within the scope of the presentinvention.

Thus, the only limitation generally on the particular restriction enzymeemployed for DNA fragmentation is that such enzyme should preferably becompatible with cloning sites present in the particular cloning vehicleemployed.

In the present disclosure, the inventors have utilized two differenttypes of cloning vehicles--an E. coli plasmid, pUC7, and abacteriophage, EMBL4--both of which are capable of introducing arecombinant fragment into an E. coli cell in a manner wherein theintroduced fragment can be transcribed by the transformed or transfected(as is the case with phage introduction) E. coli cell.

In both cases, the present inventors have chosen to utilize a Bam HIcloning site present in both the plasmid and bacteriophage DNA sequence.The recognition site for Bam HI, a six-base pair recognition enzyme, iscompatible with the DNA ends generated by enzyme Sau 3A, a four-basepair recognition enzyme. Since Sau 3A has a four-base specificity, therewill necessarily be more recognition sites for Sau 3A in any given DNAsegment. Therefore, the use of Sau 3A, or other four-base pair specificenzymes, to generate "partially" restricted DNA will provide a greaterrandomization of DNA fragments. Thus, the use of four-base pair specificenzymes are preferred, and in particular, where a Bam HI cloning site isutilized, the use of Sau 3A is preferred due to the compatibility of Sau3A-generated fragments with Bam HI sites.

In one embodiment, the present inventors have employed total Bam HIdigestions of P. haemolytica DNA for cloning into the Bam HI site ofEMBL4. The successful generation of recombinant cells which expressindividual P. haemolytica antigens demonstrates that total restrictiondigestions may also be employed where desired, at least where the enzymeemployed has a 6-base pair specificity which can thus generate fragmentswhich are sufficiently long to code for antigenic protein sequences.However, partial Sau 3A-restricted DNA has also been successfullyemployed in the context of EMBL4 to generate P. haemolytica clone banks.

Therefore, the present invention demonstrates the successful developmentof P. haemolytica DNA clone "banks" constructed in E. coli, using eithera recombined E. coli-specific bacteriophage or plasmid. As referred toherein, a P. haemolytica clone "bank" is defined as a compositioncomprising a population or plurality of individually transformed (ortransfected or infected as the case may be) recombinant cells, whereinthe transformed or transfected population has been transformed ortransfected with a recombinant vector which has individual P.haemolytica DNA fragments ligated thereto. As referred to herein, theterm "transforming" shall be used to include other types of DNAintroductions into cells and is meant to include, for example,transfection and infection.

As will be understood by those of skill in the art, such clone banksserve many useful purposes, for example, as a starting population forthe selection of individual clones therefrom which clones provideindividual P. haemolytica antigens and other proteins.

Selection of transformed E. coli cells from clone banks, which cellsproduce a selected P. haemolytica pasteurellosis-associated antigen, isachieved most preferably by, first, culturing cells from theunfractionated clone bank population on a solid support medium, forexample, on agar-containing plates, in a manner to provide for thegrowth of separated, individual clonal colonies on the surface of themedium. One then typically fashions an imprint of the clonal coloniesonto a suitable membrane support, for example, a nitrocellulosemembrane, which membrane is then subjected to suitable immunoreactionwith sera from a pasteurellosis-infected cow. Thus, clones which arepositive for the production of Pasteurella antigens are selected bymeans of a label attached either to the reactive sera or to a secondantibody which is specific for bovine sera.

Through practice of the foregoing recombinant techniques, the presentinventors have identified two P. haemolytica antigens in addition tothose antigens identified by supernatant fractionation. These clonedantigens have been identified as having molecular weights ofapproximately 66K and 53K Daltons, as ascertained by polyacrylamide gelelectrophoresis and immunoblot analysis. It will be appreciated that thesizes of P. haemolytica proteins which are produced by recombinant E.coli may be different than corresponding proteins naturally produced byP. haemolytica itself. This may be due to differences inpost-translational protein modification by the respective cells or,alternatively, may be the result of the cloning of a partial codingsequence which is an antigenic functional equivalent of the entireprotein.

Therefore, the present invention is directed to compositions whichinclude at least one of the eleven P. haemolytica antigens (i.e.--one ofthe seven supernatant protein groups I-VII or one of the two recombinantproteins), or antigenically functional equivalents thereof identified bythe foregoing procedures, and further to pharmaceutical compositionswhich include a pharmaceutically acceptable excipient or adjuvant.Suitable pharmaceutical carriers include inert solid diluents orfillers, a sterile aqueous solution, various organic solvents,emulsifying or suspending agents, or aqueous diluents such as water,ethanol, propylene glycol, glycerin, or combinations thereof.

For parenteral administration, the antigens may be formulated in sesameor peanut oil, aqueous propylene glycol, or in sterile aqueoussolutions. Such solutions are typically suitably buffered if necessaryand the liquid diluent first rendered isotonic with sufficient saline orglucose. Additionally, stabilizers in the form of, for example, sorbitolor stabilized gelatin may be included. These particular aqueoussolutions are particularly well suited for intramuscular andsubcutaneous injection, as is generally preferred for vaccination usingantigenic preparations.

However, to increase the potential antigenicity, and thereby improve theperformance of antigen-containing pharmaceutical preparations, one mayadditionally desire to include various immunoadjuvants, such as thewater-in-oil emulsion developed by Freund. The basic ingredients oflight mineral oil (Bayol) and emulsifying agents mixtures such asArlacel (A or C) are available commercially. The antigens are emulsifiedin either solutions or suspensions of the immunogen (incomplete Freund'sadjuvant). Moreover, the addition of mycobacterium (M. Butyricum, M.tuberculosis) in small amounts to the suspension (complete Freund'sadjuvant) leads to a further enhancement of the immunogenicity of thepharmaceutical vaccines made in accordance with the present invention.

In still further embodiments, the present invention is directed toantibody compositions, both polyclonal and monoclonal, havingspecificity for one or more of the eleven antigens identified by thepractice of the present invention. In general terms, polyclonalantibodies having affinity for one of the selected P. haemolyticaantigens are obtained by first immunizing an immunocompetent Mammal withthe selected antigen to obtain an immune response by the mammal,obtaining immune serum from the immunized mammal, and fractionating theserum to provide the antibody. Alternatively, the mammal may beimmunized with a less purified P. haemolytica protein preparation, forexample, Pasteurella supernatants, and the desired specific antibodyisolated from the resultant antibody mixture by, for example, adsorptionto antigen-Sepharose columns by techniques known in the art.

Monoclonal antibodies to Pasteurella antigens may be obtained by thewell-known technique of hybridoma development as detailed, for example,in U.S. Pat. No. 4,196,265, incorporated herein by reference. Ingeneral, the technique involves fusing spleen cells of a rodent withmyeloma cells from the same rodent species, wherein the rodent providingthe spleen cells has been immunized with the selected Pasteurellaantigen; culturing the fused cells in a selective medium; testing forthe presence of antibodies which are capable of immunocomplexing withthe selected antigen; culturing cells producing antibodies which arecapable of reacting with the selected antigen; and obtaining antibodiesfrom the culture supernatant of the cells.

Accordingly, a method is additionally provided whereby passive immunityto P. haemolytica infection is conveyed to a cow by administering to thecow a composition which includes a therapeutically effective amount ofantibodies having specificity for one or more of the Pasteurellaantigens identified by the present invention. Thus, high-risk cattle,for example, cattle being shipped, may be administered P.haemolytica-specific antibody compositions in the form of immuneglobulins as a temporary prophylaxis to pasteurellosis, or as atreatment during early stages of the disease.

In further embodiments employing Pasteurella-directed antibody orantigen compositions, methods are provided for detecting the presence ofsuch antigens or antibodies, as the case may be, in the serum ofsuspected infected cattle as a means of diagnosing the disease. In thecase of antibody detection, the method includes obtaining a biologicsample suspected of containing antibodies, such as serum, blood, pleuralfluid, or tissue samples, from the cow; contacting antibodies from thebiologic sample with a selected antigen under conditions which willallow for the formation of specific antigen-antibody immunocomplexes;and detecting the formation of an antibody-antigen immune complexbetween the antibody and antigen, the formation of such a complex beingindicative of the presence of the selected antibody in the sample.Preferably, the immunocomplex formation is detected by means of a labelas is known in the art. For antigen detection, the general methodologyis the same except that the biologic sample is contacted with anantibody-containing preparation.

Diagnostic kits for diagnosing Pasteurellosis in a cow are also providedwherein such kits include one or ore of the P. haemolytica antigens, orantibodies having pecificity therefor, together with a suitableimmunoetection reagent, for example, a radioactive or enzymatic ligandattached to the antigen or antibody or to a second antibody havingspecificity for the first.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Immunogenic Pasteurella Supernatant Proteins. Immunoblot offresh P. haemolytica cell-free supernatants separated on 7.5%SDS-polyacrylamide gel, electroblotted to nitrocellulose and probed witha 1/1000 dilution of convalescent bovine serum. Lanes: M, prestainedmolecular weight markers with sizes given in kilodaltons; 1, PHL101; 2,ATCC14003; 3, 194; 4, 195; 5, 199.

FIG. 2. Production of Supernatant-Specific Antibodies in a SteerImmunized with P. haemolytica Supernatant Proteins. Nitrocellulosestrips carrying separated P. haemolytica supernatant proteins wereprobed with 1/1000 dilutions of bovine serum from the following sources:C, convalescent animal; FCS, fetal calf serum,: P1 and P2, preimmunetest animal before immunization; 1 through 8, test animal one througheight weeks following immunization with samples being drawn weekly.

FIG. 3. Partial Separation of Supernatant Protein Antigens byElectroelution. P. haemolytica supernatant proteins were separated on a7.5% SDS-polyacrylamide slab gel. The gel was cut into horizontal slicesand proteins electroeluted from these slices as described. Elutedproteins were analyzed by SDS-PAGE and immunoblotting. Lanes: M,kilodaltons; S. P. haemolytica supernatant; 1, 80-100 kD slice; 2, 60-80kD slice; 3, 40-60 kD slice. The immunoblot was probed with a 1/1000dilution of convalescent bovine serum.

FIGS. 4A and 4B. Plasmid Clone pSH200 Encodes a 66 KilodaltonPasteurella Antigen in E. coli. Immunoblots of E. coli and P.haemolytica whole cell lysates and a P. haemolytica supernatantpreparation were probed with: (FIG. 4A) 1/1000 dilution of convalescentbovine serum, or (FIG. 4B) antigen eluted from E. coli cells carryingpSH200. Lanes: a, KK2186 (pUC7); b, KK2186 (pSH200); c, PHL101 wholecell lysate; d, PHL101 cell free supernatant; M. molecular weightmarkers with sizes shown in kilodaltons.

FIG. 5. Recombinant Bacteriophage Lambda SH-20 Encodes a 55 KilodaltonAntigen Recognized by Convalescent Bovine Serum. Immunoblot of phagelysates grown on E. coli NM538: V, vector EMBL4; 20, recombinant 20.Lane M contains prestained molecular weight markers with sizes shown inkilodaltons. The blot was probed with a 1/1000 dilution of convalescentbovine serum.

FIG. 6. Western blot analysis of antigens produced by recombinant lambdaphages in E. coli. M, prestained protein molecular weight markers withsizes given in kilodaltons; 1, EMBL4; 2, lambda sh20; 3, lambda sh132;4, lambda sh127; 5, P. haemolytica supernatant. Phage lysates containing10⁷ -10¹⁰ pfu/ml were electrophoresed on SDS-polyacrylamide gels, thegels electroblotted to nitrocellulose and the blots probed with immunebovine serum. Lambda sh20, lambda sh127, and lambda sh132 are exemplaryof the three types of recombinant phages that were detected byimmunoscreening. Lambda sh127 is a member from the Bgl II library thatproduces the same 66 KD antigen as pSH200. Lambda SH20 (Bam HI library)encodes a 55 KD antigen, while the 105 kD antigen of lambda sh132 (BglII library) corresponds to supernatant antigen I.

FIGS. 7A and 7B. Genetic and physical map of recombinant phase andplasmids containing the ptx gene. E, Eco RI; H, Hinc II; P. Pst I; B,Bgl II; A, Ava I; C, Cla I. Restriction enzyme mapping of lambda sh 132indicated that the recombinant phage contained two BglII sites andsuggested that the insert was derived from three chromosomal Bgl IIfragments. The insert also contained a single Eco RI site. The twoconstituent Eco RI fragments (17.6 and 1.2 kb) were subcloned into theEco RI site of the lacZ filamid, pBS, and the resulting plasmids,pSH207, pSH209 and pSH210, were tested for their ability to produce the105 kD antigen. Strains carrying these and other deletant plasmids (FIG.7A) were screened by Western blot analysis of whole cell E. coli lysates(FIG. 7B). Plasmids pSH207 and pSH209 produced the antigen but pSH210did not. To further delimit the ptx gene, simple deletants and subcloneswere constructed from pSH207 and then tested for antigen production.These mapping experiments identified the 5.2 kb Ava I-Eco RI fragment ascontaining the ptx gene and also showed that the 3.9 and 6.4 kb Bgl IIfragments of the phage insert were contiguous within the P. haemolyticachromosome.

FIG. 8. Transcriptional frame of the ptx gene and overexpression of thePTX protein under lac transcriptional control. Comparison of the amountof 105 kD antigen produced by pSH207 and pSH209 (FIG. 7A and 7B)suggested that ptx expression was influenced by vector sequences,particularly by transcription from the lac promoter on pBS. This wasverified by comparing the amount of antigen produced by each plasmid inthe presence and absence of IPTG. Whole cell lysates were prepared andanalyzed by Western blotting for pBS (lane 1), pSH207 (lanes 2, 3 and 4)and pSH209 (lanes 5, 6 and 7) from: 1, 2 and 5, overnight culture; 3 and6, log-phase culture, uninduced; 4 and 7, log-phase culture, 3 hr.induction with 0.5 mM IPTG. M, prestained protein molecular weightmarkers with sizes shown in kilodaltons. The immunoblot illustrates thatptx expression from pSH209 is increased at least 10-fold in the presenceof IPTG, while pSH207 expression is not affected by induction of the lacpromoter. The production of antigen by pSH207 does, however, indicatethat the Pasteurella ptx promoter is transcribed in E. coli, albeit at acomparatively low level.

FIGS. 9A-9L. The Nucleotide Sequence and Corresponding Amino AcidsSequence of the ptx Gene and the PTX Protein. Shown is the nucleotidesequence of the ptx gene and its encoded product, the 105K Daltonantigen or PTX protein. The nucleotide sequence was determined usingsingle stranded templates from subclones in M13 and pBS vectors, and theT7 DNA polymerase, Sequenase kit of United States Biochemicals(Cleveland, Ohio). The sequence was then analyzed by subjecting it tothe Pustell DNA Sequence Analysis Program of InternationalBiotechnologies, Inc. (New Haven, Conn.).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is disclosed in terms of the two generalapproaches employed by the inventors to identify and isolate P.haemolytica antigens which are recognized by immune sera frompasteurellosis-infected cattle. The first approach involves theisolation of antigens identified in P. haemolytica cell-freesupernatants, which the second approach utilizes recombinant DNAtechnology to provide cells which produce individual P. haemolyticaantigens.

The supernatant approach is based on the premise that P. haemolyticabacterins inoculation fails to provide an effective immunization againstpasteurellosis because such bacterins do not contain all of theantigenic elements necessary to provoke an appropriate immune response.It is believed that antigenic elements present in cell-free supernatantscan serve to supply those elements which are missing from bacterinpreparations. The fact that there are proteins present in thesupernatant which are recognized by pasteurellosis-derived antiserademonstrates that the supernatant proteins identified by the presentinventors are in fact present during active infections and areimmunogenic. Moreover, it is known that cell-free supernatants containthe leukotoxin activity. Therefore, a key feature of the presentinvention is the use of P. haemolytica cell-free supernatants as asource of antigens which may be employed alone, or together with P.haemolytica bacterins, to immunize cattle.

A. Identification of P. haemolytica Supernatant Antigens

As noted above, the supernatant approach involves the identification ofantigenic P. haemolytica polypeptides present in a cell-free P.haemolytica culture supernatant. In general, this approach involves,first, culturing P. haemolytica bacteria to produce a culturesupernatant which includes individual P. haemolytica polypeptides. Afterremoving the cells from the culture supernatant, for example, bycentrifuging out the cells and pouring off the supernatant, theresultant cell-free supernatant is subjected to polyacrylamide gelelectrophoresis to fractionate the proteins according to their molecularweights.

Next, the antigens of the present invention are identified by theirability to be recognized by antisera from pasteurellosis-infectedcattle. Typically, and most conveniently, such identification isaccomplished by immunoblotting which involves transferringgel-fractionated polypeptides onto a nitrocellulose sheet, andsubjecting the protein-imprinted sheet to immuno-reaction withPasteurella-induced antisera. The antigens of the present invention maythen be identified by means of a label associated with antibodymolecules of the antisera or with a second antibody, which label servesto identify the gel migration distance, and hence, the molecular weight,of the antigens. The following example, Example I, demonstrates theforegoing general embodiment in more specific terms as practiced by theinventors.

EXAMPLE I Isolation of P. haemolytica Supernatant Antigens

1. Bacterial Strains, Media, and Bovine Sera. Pasterurella haemolyticastrain PHL101 was obtained from Dr. G. H. Frank (USDA, Ames, Iowa).Strain ATCC 14003 was obtained from the American Type Culture Collection(Rockville, Md.). Other P. haemolytica strains were isolated from thelungs of cattle exhibiting symptoms of pasteurellosis.

Pasteurella strains were routinely cultivated on Blood Agar Basecontaining 5% sheep blood (Scott Laboratories, Fiskeville, R.I.) andwere grown in Brain Heart Infusion (BHI) broth (Difco Laboratories,Detroit, Mich.) or RPMI 1640 medium (Sigma, St. Louis, Mo.) at 37° C.

Bovine sera used for immunodetection were isolated from whole bloodsamples drawn from adult animals and calves. Only cattle which exhibiteddiscrete symptoms, such as sniffling, wheezing, respiratory distress,cough, fever and nasal drainage, were selected as antisera donors. Also,it was found that cattle convalescing from the disease served as goodsources for Pasteurella-reactive antisera.

2. Preparation of Whole Cell Lysates and Cell-Free Supernatants forProtein Analysis.

Cells were grown to a density of about 10⁸ to 10⁹ cells/ml in BHI andthen harvested by centrifugation 10 minutes at 12,000×g. For whole celllysates, the cell pellet was resuspended in a 1/14th volume of 2× SDSgel loading buffer (125 mM Tris, pH 6.8, 20% glycerol, 10%B-mercaptoethanol, 4.5% SDS, 0.005% bromophenol blue) and boiled 5minutes before use (see, e.g., Silhavy, et al., Experiments with GeneFusions, Cold Spring Harbor, 1984). Cell-free supernatants were preparedfrom the BHI supernatant or from a similar supernatant derived fromcells that had been diluted 1/10 in RPMI 1640 and then grown to 10⁹cells/ml. In either case, the supernatant was passed through a sterile0.22 um filter and the filtrate used or stored frozen for furtheranalysis.

Frequently, culture supernatants were concentrated with polyethyleneglycol 6000 (PEG), as follows. The filtered supernatant was enclosed indialysis tubing (exclusion limit 15,000 daltons), then was completelycovered with PEG, and allowed to stand overnight at 4° C. Theconcentrated supernatant was removed from the dialysis tubing,transferred to clean tubing and then dialyzed 16 to 24 hours, at 4° C.,versus 100 volumes 10 mM Tris, pH 7.5. Following dialysis, theconcentrate was lyophilized and the protein resuspended in 10 mM Tris at0.01× the starting volume.

3. Immunodetection of Proteins.

Whole cell lysates and cell-free supernatants were electrophoresed on7.5% running, 3% stacking sodium dodecyl sulfate (SDS)/polyacrylamidegels as described by Laemmli (1970), Nature, 227:680. Supernatantsamples were mixed with a 1/3 volume of 3× SDS gel loading buffer (2×described previously), and all samples were boiled five minutes beforebeing loaded onto a gel. For direct visualization of proteins, gels werestained either with Coumassie brilliant blue (Laemmli, supra.) or withsilver stain reagents (Merril, et al. (1981), Science, 211:1437) asdirected by the supplier (BioRad, Richmond, Calif.).

Protein antigens recognized by immune bovine serum were detected inSDS/polyacrylamide gels using the western blotting technique of Towbin,et al. (1979), Proc. Natl. Acad. Sci., U.S.A., 76:4350, and as follows.After electrophoresis, a 7.5% SDS/polyacrylamide gel was soaked for 60minutes at room temperature in 200 ml 1× Electroblot Buffer (25 mM Tris,pH 8.3, 192 mM glycine) containing 4M urea, 2 mM Na₂ EDTA, and 0.1 mMdithiothreitol (DTT). The gel was rinsed twice with fresh ElectroblotBuffer, placed onto a sheet of 0.45 um nitrocellulose (Schleicher andSchuell, Keene, N.H.), and then sandwiched between several sheet ofWhatman 3 MM filter paper. The entire assembly was placed betweenblotting electrodes, with the nitrocellulose sheet facing the anode, andlowered into a chamber containing precooled 1× Electroblot Buffer. Acurrent of 0.02 amperes was applied for 16 to 20 hours at 4° C., causingthe proteins to be transferred from the gel onto the nitrocellulosesheet.

The nitrocellulose sheet, or blot, was preincubated for 60 minutes at37° C. in 100 ml 1× TBS (10 mM Tris, pH 7.6, 0.9% NaCl) containing 2%w/v nonfat dry milk to reduce non-specific binding of antibodies to thesheet.

Bovine serum was then added (usually to yield a 0.002 to 0.001 dilution)and the incubation was continued for 2 hours at 37° C. The blot was thenwashed five times in 100 ml 1× TBS for a total of 30 minutes to removeany unbound antibody.

Immune complexes were detected using biotin conjugated goat anti-bovineIgG and horseradish peroxidase (HRP) conjugated steptavidin (Kirkegaardand Perry Laboratories, Inc., Gaithersburg, Md.), (Guesdon, et al.,(1979), J. Histochem. Cytochem., 27:1131): the blot was incubated for 2hours in 100 ml 1× TBS, 2% milk containing 0.1 mg biotin-anti-bovine IgGat 37° C., washed five times in 100 ml 1× TBS for thirty minutes, thenincubated 60 minutes at 37° with 100 ml 1× TBS, 2% milk containing 0.05mg HRP-streptavidin, and washed again five times. Bound HRP was detectedby incubating the nitrocellulose blot with 100 ml 50 mM Tris, pH 7.5,0.2M NaCl containing 60 ul hydrogen peroxide (30% solution) and 0.5 mgof the chromogenic substrate, 4-chloro-1-napthol (Hawkes, et al., 1982).Color development was complete within thirty minutes at roomtemperature.

FIG. 1 is illustrative of a typical immunoblot of various P. haemolyticastrains. Demonstrated in the figure are various P. haemolyticasupernatants which have been first subjected to SDS-polyacrylamide gelfractionation on a 7.5% gel. After electrophoresis and electroblottingas described above, the resultant blot was probed with a 1/1000 dilutionof convalescent bovine serum. Prestained molecular weight markers wererun in lane M, from which the molecular weights, in kilodaltons, wereassigned and placed along the left-hand side of the figure. Cell-freesupernatant proteins from various P. haemolytica strains were run on thegel as follows: lane 1, PHL101; lane 2, ATCC 14003; lanes 3-5, variousother strains also isolated from naturally-infected cattle (strains 194,195, and 199, respectively).

A semi-logarithmic plot of standard marker migration versus their knownmolecular weights was constructed. By comparing the migration of thevarious antigens to the molecular weight plot in this and numeroussimilarly performed experiments, molecular weight ranges have beenassigned to the individual antigens, or antigen groups. The followingtable, Table I, is a compilation of those findings:

                  TABLE I    ______________________________________    Molecular Weights of P. haemolytica Antigens                   Apparent                   Molecular Weight                               Reference    Antigen Group  Range       Weight    ______________________________________    I                     98-140K  105K    II                    86-110K  90K    III                  76-85K    76K    IV                   73-82K    73K    V                    63-71K    65K    VI                   42.5-45K  43K    VIIa                           35K                         29-35K    32K    c                              29K    ______________________________________

Thus, referring to FIG. 1 in particular, there can be seen a series ofP. haemolytica antigens, or antigen groups, which have migrated to aposition which corresponds generally to their approximate molecularweights. Antigen I was found to exhibit an apparent molecular weightrange of between 98 and 140 kilodaltons, with a reference weight ofabout 105 kilodaltons. The "bowing-out" labeling and intensity of theprotein banding configuration of Antigen I suggested that it is presentin relatively higher concentrations in the Pasteurella supernatants, andthat the protein(s) is particularly antigenic.

A second antigen, Antigen II, migrated to a position corresponding toabout 86-110 kilodaltons, with a reference weight of about 90kilodaltons.

A third antigen, Antigen III, migrated to a position corresponding toabout 76 to 85 kilodaltons, with a reference weight of approximately 76kilodaltons.

A fourth antigen, Antigen IV, migrated to a position corresponding toabout 73 to 82 kilodaltons, with a reference weight of about 73kilodaltons. Thus, Antigens III and IV appear generally as a distinctivedoublet, with Antigen III running slightly behind Antigen IV.

A fifth antigen, Antigen V, migrated to a position corresponding toabout 63 to 71 kilodaltons, with a reference molecular weight of about65 kilodaltons.

A sixth antigen migrated to a position correspond to about 42.5 to 45kilodaltons, with a reference molecular weight of about 43 kilodaltons.

Three additional antigens were found to migrate to positionscorresponding to about 35, 32 and 29 kilodaltons. These antigens wereassigned the designations Antigen's VIIa-c, respectively, in that, ascan be seen, only one member of the group has been seen in any one P.haemolytica strain. Thus, it is believed that the three antigenicspecies represent proteins which are modified, e.g., glycosylated, todiffering degrees, or differ in terms of amino acid sequence.

4. Immunization of Calves and Rabbits with P. haemolytica SupernatantProteins.

Both calves and rabbits were injected with P. haemolytica proteins todemonstrate that the proteins were immunogenic. Rabbit care, inoculationand serum isolation was performed by Bethyl Laboratories, Montgomery,Tex. Rabbits were injected, subcutaneously with 900 ug concentratedsupernatant proteins combined with 500 ul Freund's incomplete adjuvant.Animals were boosted on day 21 with 900 ug of supernatant protein inincomplete Freund's adjuvant. Rabbits were bled weekly, beginning threeweeks after the booster injection, and serum prepared: these sera weretested for their ability to recognize P. haemolytica supernatant andwhole cell lysate proteins by western blotting of lysates andsupernatants, as previously described.

Bovine experiments were performed using a twelve month old, 990 kg.,Black Angus steer, pastured in New Summersville, Tex. The animal wasinoculated subcutaneously with 200 ug P. haemolytica concentratedsupernatant linked to one ml alum adjuvant on day one and then similarlyboosted with the same mixture on day 21. Blood samples were collected inseven day intervals for eight weeks and serum was prepared. Sera weretested for the presence of, and found to contain, antibodies specific toP. haemolytica supernatant and whole cell lysate proteins by westernblotting, performed as before.

FIG. 2 is an immunoblot of P. haemolytica supernatant proteins which wascut into individual vertical strips. These individual strips were thenincubated with 1/1000 dilutions of bovine serum from the followingsources: lane C, convalescent serum (i.e.-serum from anaturally-infected, convalescent animal); lane FSC, fetal calf serum;lanes P1 and P2, preimmune test animal before immunization; lanes 1through 8, serum from test animal, collected weekly, one through eightweeks following immunization with the antigenic composition includingconcentrated, dialyzed P. haemolytica supernatant proteins.

As can be seen from FIG. 2, the antigens recognized by the convalescentserum (lane c) were found to correspond generally to the antigensrecognized by the test animal's sera. In particular, it was noted thatthe antibody titer for these particular antigens (Antigens I-VII)increased during the inoculation period, with antibodies to Antigens I,III, IV, V and VII increasing most dramatically. Thus, FIG. 2demonstrates the antigenicity of the P. haemolytica supernatant, and ofthe individual antigens I-VII, and further, the ability of thesupernatant to induce a response which is similar to, and augmentedabove, that seen in a convalescent animal.

As noted in the summary, the "reference weight" above refers to theweight which represents the inventors best estimate of a specificmolecular weight. As such, the particular antigen groups may at times bereferred to, for convenience, in terms of either the reference weight orthe antigen group designation. Such references should not be interpretedto limit the scope of the present invention to any such specificreference molecular weight and is meant to include the range as a whole.

5. Elution of Antigen-Specific Antibodies from Nitrocellulose Blots.

To examine the antigenic relationship of one protein species to another,antibodies were eluted from a nitrocellulose blot and used to probe asecond blot. Proteins separated on SDS/polyacrylamide gels weretransferred to nitrocellulose, as described above. The blot was thenincubated 10 minutes in a 0.2% solution of Ponceau S (Sigma Diagnostics,St. Louis, Mo.) to temporarily visualize the transferred proteins.Horizontal or vertical strips were cut from the nitrocellulose and thesestrips were treated with 1× TBS, 2% milk followed by primary antibodyincubation, as described above. The strips were washed three times, atroom temperature, in 100 ml 1× TBS 1% milk, for twenty minutes each andthen rinsed briefly in 100 ml 1× TBS. The bound antibodies were removedby vortexing a crumpled nitrocellulose strip in 2 ml glycine-Hcl, pH 2.5for two minutes. One ml of 0.5M K₂ HPO₄, pH 9.0 was added immediatelyand the strip was vortexed again. The eluate was aspirated from the tubeand then dialyzed for 16 hours at 4° C. versus 1× TBS. The dialysate wascentrifuged 5 minutes at 8000×g, 4° C. to pellet the milk protein, andthe clear supernatant, containing the eluted antibodies, was reserved.This solution was made 2% (w/v) in nonfat dry milk and then was used asprimary antibody to probe other protein blots, as described in theprevious section.

6. Electroelution of Pasteurella Supernatant Proteins from AcrylamideGels.

Concentrated culture supernatants from P. haemolytica wereelectrophoresed on 7.5% SDS-polyacrylamide gels in one wide, 16 cm,well. Using prestained molecular weight markers as a guide, gel slices,containing specific protein bands, were cut from the gel. Each gel slicewas immersed in SDS Gel Electrode Buffer (0.25M Tris, pH 8.3, 0.192Mglycine, 1% SDS) and the protein was eluted from the acrylamide at apower of one watt for three hours, then 3 watts for an additional hour.The apparatus was maintained at 4° C. using a circulating ice-water bathand 1 ml samples of eluted protein solution were removed at 60 minuteintervals. Aliquots of the eluted proteins were reelectrophoresed on anSDS-polyacrylamide gel to monitor recovery and purity.

FIG. 3 illustrates a typical immunoblot of proteins fractionated by gelelectrophoresis, isolated by gel electroelution, and rerun on a 7.5%SDS-polyacrylamide slab gel as follows: lane m, prestained molecularweight markers with sizes given in kilodaltons; lane a, P. haemolyticasupernatant; lane 1, 80-100 kilodalton slice; lane 2, 60-80 kilodaltonslice; lane 3, 40-60 kilodalton slice. The immunoblot was probed with a1/1000 dilution of convalescent bovine serum.

As can be seen from FIG. 3, the 80 to 100 kilodalton slice includedprimarily Antigens I-IV, the 60 to 80 kilodalton slice includedprimarily Antigens IV-VI, and the 40 to 60 kilodalton fraction was foundto include primarily Antigens VI and VII.

7. DEAE Sephadex Column Chromatography

Concentrated P. haemolytica supernatants were chromatographed over 15 cmby 150 cm² DEAE-Sephadex A25-120 columns which had been equilibratedwith 10 mM Tris, pH 7.5. A single protein species was eluted with a 0.5ml NaCl wash and collection of 20 ml fractions. This protein wassubjected to immunoblot analysis and found to include primarily AntigensI and II.

8. Gel Filtration Column Chromatography

An ammonium sulfate precipitate of P. haemolytica supernatant waschromatographed on a 125 cm by 1.75 cm² Pharmacia Sephacryl 400superfine column equilibrated with 10 mM potassium phosphate buffer, pH7.6, 0.8% NaCl, 0.05% NaN₃. Sample volumes from 2.5 to 10 ml wereapplied and chromatographed in the equilibration buffer, and 1.5 mlfractions were collected. Typically, the bulk of the antigenic materialwas found to exclude from the column, suggestive of a high molecularweight antigen complex. This complex included Antigens I-VII, in arelatively purified form relative to unfractionated supernatant.

9. Antibody-Sepharose Chromatography

An alternate, or additional approach to the purification of antigens isthrough the use of antibody-Sepharose chromatography. In general, theapproach requires the attachment of pasteurellosis-derived antisera to asuitable solid support, for example, Sepharose, and contacting theantibody-bound support with the cell-free supernatant so as to obtainbinding of specific antigens to the antibodies. Methods for bindingantibodies to affinity matrixes are well known in the art as, forexample, detailed in Methods in Enzymology, Vol. 34B. After theimmuno-complexed support is washed thoroughly to remove non-specificallybound proteins, the specifically-bound antigens are eluted to provide asubstantially purified antigen mixture. One method which may be employedfor conjugation to Sepharose is as follows:

The gel is first washed with distilled water. A ratio of approximately 1g of protein to 30 g of dry gel (dry weight equals approximately volumeof wet packed gel divided by 1.6) is utilized. To one volume of wet geladd a volume of 2M Na₂ CO₃, and stir slowly and chill at 5° C. Then add2 g of cyanogen bromide per 30 g dried gel (CNBr; dissolved in CH₃ CN at2 g/ml) to the chilled mixture and stir vigorously for 1-2 minutes. Themixture is then poured into a cooled sintered glass funnel and washedrapidly with 10-20 volumes of cold 0.1M NaHCO₃. One volume of 0.2MNAHCO₃ containing the dissolved protein is added and the mixture,stirred gently for 20 hours at 4° C. Then it is washed on a sinteredglass funnel with 10-20 volumes of 0.1M acetic acid with 0.5M NaCl, thenwith 0.1M NAHCO₃ (pH above 8.0). Then, an equal volume of ethanolamine(1M in 0.2M NaHCO₃) is added and the mixture is stirred for about 4hours. The mixture is then washed on a sintered glass funnel with 3M KClin 0.1M sodium phosphate buffer, pH 7.0, and then with starting columnbuffer.

Next, the supernatant is dissolved in, or dialyzed into, a buffer inwhich it is stable with an appropriate ionic strength to allow for theformation of an antigen-antibody complex (e.g.--0.02M phosphate buffer,0.25M NaCl, pH 7.6). It is then passed over the matrix-bound antibodyusing the same buffer. After washing the column to remove unboundmaterial, the specifically bound antigens are eluted with one of severalsolutions, for example, 0.1M acetic acid (for a low affinity antigenfollowed by 0.5M acetic acid (to elute high affinity antigens); 0.05Macid, pH 2.5 0.05M glycine-HCl buffer, pH 2.5; or 0.1M acetic acidfollowed by 6M urea. Where 6M urea is utilized, one will need to dialyzeout the urea in a step wise fashion, for example, by reducing the ureaconcentration in the dialysate in molar increments.

B. PRODUCTION OF RECOMBINANT CELLS EXPRESSING P. HAEMOLYTICA ANTIGENS

The second general overall approach employed to identify P. haemolyticaantigens involves the use of recombinant DNA technology. In particular,cells have been genetically engineered to express individual P.haemolytica antigens by transforming E. coli cells with randomized P.haemolytica DNA fragments to produce P. haemolytica clone banks throughthe use of two different types of E. coli cloning vectors--plasmid pUC7and bacteriophage EMBL4. These vectors were chosen for convenience inthat they are readily available and have been found by the presentinventors to provide suitable clone banks for practicing the invention.

Although the foregoing cloning systems have been employed by the presentinventors by way of illustration and convenience, it will be recognizedthat virtually any cloning system may be employed. For example, in thatthe cloning processes involve the cloning of bacterial sequences, itwill be appreciated that there is no general requirement that anexpression vector be employed to obtain expression of the clonedsequences, in the form of individual P. haemolytica antigen production.Due to the relative lack of complexity of bacterial gene controlsequences, at least a sufficient proportion of randomized P. haemolyticaDNA fragments will contain sequences sufficient to control theexpression of the DNA when introduced into E. coli, regardless of thecloning vector employed. However, the use of a so-called "expression"vector (i.e.--a vector having built-in gene expression capability) maybe employed to improve the percentage of recombinant clones present inthe clone bank population which are actively expressing cloned P.haemolytica sequences.

In general, recombinant cells produced in accordance with the presentinvention are made by, first, isolating P. haemolytica DNA from any ofthe various serotype A1 strains, or from strains isolated frompasteurellosis-infected cattle. By way of illustration the inventorshave employed strain PHL101. However, virtually any strain which iscapable of eliciting pasteurellosis may be employed.

After isolation of the bacterial DNA, it then must be fragmented,preferably by a random fragmentation method. As discussed previously,random fragment generation provides a P. haemolytica DNA fragmentpopulation such that virtually every P. haemolytica gene is representedwithin the population. Moreover, even the most unique gene will bepresented to an extent sufficient to provide a protein-expressing clonebank wherein the expression of virtually every transcriptionally activeP. haemolytica gene is represented within the E. coli clone bankpopulation.

The random DNA fragmentation method employed herein is partialrestriction enzyme digestion. Partial restriction enzyme digestion is apreferred means of fragmentation because the random fragmentsso-generated will typically have restriction enzyme-produced "stickyends" which may be readily annealed and ligated to correspondinglygenerated "sticky ends" of cloning vectors. However, other methods maybe employed to generate random DNA fragments which are suitable. Forexample, the DNA may be mechanically or chemically sheared, through theuse of a Waring blender, French pressure press sonication, passagethrough a syringe needle or chemical cleavage, to provide randomfragments of a selected size. However, appropriate "linker" sequencesmust be ligated to sheared DNA in order to ligate the sequences with thecloning vector. Such alternate techniques are well known in the art andthus will not be presented in greater detail herein.

There is no actual requirement that P. haemolytica DNA be randomlyfragmented in that there is no requirement that full protein codingsequences be cloned and expressed in the host, only that antigenicallyfunctional equivalents be expressed. In one embodiment, P. haemolyticaDNA is fragmented through the use of total restriction digestion. Suchfragments are not random in that, with total digestion, virtually everyrestriction recognition site with the DNA molecule will be recognizedand cleaved by the selected enzyme. Thus, the DNA is reproduciblycleaved to reproducibly generate discrete fragments. Such fullyrestriction enzyme digested P. haemolytica DNA has been used tosuccessfully construct clone banks which are capable of providingrecombinant clones in accordance with the invention. However, it will beappreciate that where total restriction digestion is employed as afragmentation method, it is preferably to employ an enzyme having a moreselective sequence specificity (e.g.--six base pair specificity asopposed to four base pair specificity) in that such enzymes will havefewer recognition sites within any given DNA sequence, and hence, longerfragments will be generated. Typically, the longer the fragmentsso-generated, the greater the proportion of fragments that containsequences coding for antigen determinants.

After the DNA has been fragmented, it is then inserted into the cloningvector, by ligation of the fragments to suitably cleaved vector DNA. By"suitably cleaved" is meant that the cloning vector DNA must berestriction enzyme cleaved at a suitable recombinant site within thevector. Determination of appropriate sites for any given vector is wellwithin the skill of the art and may be determined, for example, fromspecification data supplied with the vector, when obtained fromcommercial sources, or from knowledge of the restriction enzyme map ofthe vector. Typically, a site is chosen which, when cleaved, serves toeliminate a particular genetic advantage provided by the vector to thehost when the host is transformed. For example, cloning vectors maytypically have drug resistance genes which, when left intact, confer aparticular drug resistance to the host. However, when cleaved in amanner to receive inserted fragments to be cloned, the drug resistancegene is interrupted and no drug resistance is conferred to the host. Inthis manner, successfully transformed cells may be selected by selectingfor those cells which don't display the particular drug resistance.

An alternative to the use of drug resistant markers is the use ofcloning sites within other genes present within the vector, which genes,when intact, produce a detectable product, but, when cleaved to acceptan inserted fragment, fail to produce the product. For example, many E.coli plasmids are constructed to contain sequences of the LacZ gene,which produces B-galactosidase when intact, but fails to produce thisenzyme when a sequence has been inserted therein. Thus, successfultransformants are selected on the basis of B-galactosidase production.

After construction of recombinant vectors, the vectors are used totransform an appropriate host. In a preferred embodiment, the host is anE. coli cell of a type which is compatible with the selected vectortype. However, although the present invention is disclosed in terms ofE. coli host/vector systems, other host/vector systems are known in theart and may be employed where desired. For example, numerous eukaryotichost/vector systems are known in the art (for example, see Okayama etal. (1983), Mol. Cell. Biol., 3:280, for a description of a suitableeukaryotic expression vector derived from SV-40). Such systems aresuitable for use in constructing recombinant cells in accordance withthe present invention.

Transformation of host cells by the recombined vector is achieved usingstandard procedures known in the art. For example, where plasmid vectorsare employed, transformation is typically achieved by permeabilizingcompetent cells with calcium and contacting the permeablized cells withthe recombinant vector DNA. Where bacteriophage vectors are employed,one may additionally choose to package the recombinant phage with phagecoat proteins, which affords direct transformation capability throughcell infection with a resultant increase in transformation efficiency.

Once the cells are successfully transformed with the recombinant vectorDNA, they are plated to provide individual recombinant clonal coloniesor plaques, a selected proportion of which are actively producing P.haemolytica proteins. Moreover, a portion of these translationallyactive transformants will be actively producing P. haemolytica antigens.Thus, isolation of recombinant cells in accordance with the presentinvention requires the identification and selection of those transformedcells which produce P. haemolytica proteins, or their antigenicequivalents, that are recognized by pasteurellosis-induced antiserum.Typically, this identification is accomplished by testing each of therecombinant cells with the antiserum to identify clonal colonies orplaques which positively react and which positive reaction is indicativeof P. haemolytica antigen production.

Once positive clones are selected, the antigens produced by the selectedrecombinant clones are isolated by, first, culturing the recombinantcell in a suitable media, and, if necessary, stimulating the vectorpromotor which carries the P. haemolytica gene to a state of activetranscription. After plateau phase has been achieved, the cells areharvested, lysed by sonication, the debris centrifuged out, and the P.haemolytica antigen isolated by chromatography on a gel exclusion matrixor polyacrylamide gel.

The following example, Example II, demonstrates a specific embodiment ofthe foregoing general embodiments, and illustrate the successfuldevelopment of P. haemolytica clone banks in E. coli hosts, using bothplasmid and phage vectors, and further demonstrates the identificationand isolation of transcriptionally active recombinant clones from suchclone banks, which clones produce selected individual P. haemolyticaantigens.

EXAMPLE II Construction and Identification of Recombinant CellsProducing P. haemolytica Antigens

1. Bacterial Strains

The P. haemolytica strains employed were as described for Example I. TheE. coli strains used were KK2186, NM 538 and NM 539, as described byFrischauf et al. (183), J. Mol. Biol., 170:827. E. coli strains weregrown on LB media, with the addition of 100 ug/ml ampicillin, whennecessary, or one mg agar medium supplemented with 125 ug/ml FX-Gel or0.03 mM IPTG, as described by Maniatis, et al. (1982), MolecularCloning, Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y.

2. Preparation of P. haemolytica Restriction Fragments for Constructionof a Genomic Library.

Chromosomal DNA was prepared from P. haemolytica strain PHL101 by thelysozyme-Triton lysis method described by Davis, et al. (1980), AdvancedBacterial Genetics, Cold Spring Harbor Laboratory, N.Y. Briefly, a 100ml late stationary phase culture of PHL101, grown in BHI, was harvestedby centrifugation 10 minutes at 12,000×g. The pellet was resuspended in2 ml 15% sucrose, 50 mM Tris, pH 8.5, 50 mM Na₂ EDTA, containing 1 mg/mlfresh lysozyme. The cells were incubated 60 minutes at room temperature,then 2 ml 0.1% Triton X-100 (Sigma, St. Louis, Mo.), 50 mM Tris, pH 8.5,50 mM Na₂ EDTA was added. The lysate was incubated 30 minutes more atroom temperature, then 40 ul 10 mg/ml RNAse A were added and theincubation was continued for 45 minutes at 37° C.

The resulting crude lysate was used to form a six ml ethidiumbromide-CsCl density gradient (6 ml lysate, 6 g CsCl, 0.6 ml 10 mg/mlethidium bromide) (Clewell and Helinski (1972), J. Bacteriol.,110:1135). The gradient was centrifuged 18 hours, at 60,000 rpm in a70.1 Ti rotor. The chromosomal DNA band was located using a long waveultraviolet light source and was removed from the gradient with a needleand syringe. This DNA was then subjected to a second cycle ofcentrifugation through a fresh gradient and the chromosomal fractionreisolated. The ethidium bromide was removed by extraction with an equalvolume of isopropanol and the resulting DNA solution was dialyzed 16hours at 4° C. versus 100 volumes 1× TE Buffer (10 mM Tris, pH 8.0, 1 mMNa2EDTA).

For library construction, the PHL101 chromosomal DNA was digestedpartially with Sau 3A or completely with Bam HI, as follows. 250 ugPHL101 chromosomal DNA was digested with 12 units Sau 3A (BRL, Bethesda,Md.) in a total volume of 3.0 ml 6 mM Tris, pH 7.5, 50 mM NaCl, 6 mMMgCl₂ for one hour at 37° C. The reaction was terminated by theadditional of 0.04 volume 0.5M Na₂ EDTA. The restriction fragments weresize-fractionated by centrifuging half of the digest through a 10 to 40%linear sucrose gradient (1M NaCl, 20 mM Tris, pH 8.0, 5 mM Na₂ EDTA), asdescribed by Maniatis, et al., supra (1982). The gradients werecentrifuged 24 hours at 26,000 rpm, 20° C., using a SW27 swinging bucketrotor.

The bottom of the tube was punctured, 0.5 ml fractions were collected,and aliquots were analyzed by electrophoresis through a 0.5% agarosegel, 18 hours at 30 volts, using a TBE (89 mM Tris, 89 mM boric acid, 2mM Na₂ EDTA) buffer system. Fractions containing fragments ranging from5-10 kilobase (kb) and 10-20 kb in size were pooled separately, dialyzedversus TE and then precipitated by the addition of 0.1 volume 3M NaAcand 3 volumes cold 100% ethanol. After incubating 20 minutes on ice, theDNA fragments were collected by a 10 minute centrifugation in anEppendorf centrifuge, washed once with cold 70% ethanol and then driedand resuspended in 100 ul 1× TE. Fragments were stored at -20° C.

When Bam HI fragments were required, 20 ug PHL101 chromosomal DNA weredigested with 40 units Bam HI (BRL, Bethesda, Md.), in 100 ul 20 mMTris, pH 8.0, 100 mM NaCl, 7 mM MgCl₂, for 2 hours at 37° C. Thereaction was terminated by heating 10 minutes at 65° C. and the digestwas stored at -20° C.

3. Construction of a P. haemolytica Plasmid Library in E. coli.

The E. coli cloning vector, pUC7 was selected for construction of a P.haemolytica Sau 3A fragment library. The vector carries the pBR322origin of replication, ampicillin resistance gene and a portion of theLac Z (B-galactosidase) gene with the M13mp7 multiple cloning site(Messing, et al. (1981), Methods in Enzymology, 101:10). Insertion of aSau 3A into the Bam HI site on the vector interrupts the Lac Z gene andcauses the loss of B-galactosidase activity.

Plasmid pUC7 DNA was prepared from E. coli strain KK2186, carrying theplasmid, as described for the preparation of Pasteurella chromosomalDNA, except that the bacteria were grown in LB broth containing 100ug/ml ampicillin, and that the plasmid, not chromosomal DNA band, wasremoved from the CsCl gradient. 175 ug pUC7 DNA was digested with 20units Bam HI in 700 ul 20 mM Tris, pH 8.0, 100 mM NaCl, 7 mM MgCl₂, at37° C. for two hours. The reaction was heated 10 minutes at 65° C., then2 units alkaline phosphatase (Boehringer-Mannheim, Indianapolis, Ind.)were added and incubation continued for 45 minutes at 37° C. Thereaction was again heated for 10 minutes at 65° C.

Linear pUC7 molecules were purified by electrophoresing the digestmixture on a 5% polyacrylamide gel (19 acrylamide:1 bis, 1× TBE) 3 hoursat 15 volts/cm. The gel was stained with a 2% methylene blue solutionand the DNA band was located and excised from the gel. A glass rod wasused to crush the gel slice into a fine paste and the paste wassuspended in 2 ml Extraction Buffer (10mM Tris, pH 8.0, 50 mM NaCl, 10mM Na₂ EDTA). This slurry was incubated 16 to 24 hours at 37° C. andthen spun through a 1 cm glass wool plug, 10 minutes, 2000×g, toseparate the DNA solution from the acrylamide. The DNA solution wasextracted once with TE-saturated phenol, extracted three times withanhydrous ethyl ether and then ethanol precipitated by addition of 0.1volume 3M NaAc plus 3 volumes cold 100% ethanol and incubation for 20minutes on ice. The DNA was collected by centrifugation 10 minutes in anEppendorf centrifuge, the pellet was dried, and then resuspended in 500ul TE to a final concentration of 20 ug/ml.

Bam HI-linearized pUC7 DNA was mixed with a portion of the PHL101 5-10kb Sau 3A partial digest pool and ligated as follows. 1.25 ug pUC7 wascombined with 2 ug of the pooled Sau 3A fragments and ethanolprecipitated. The DNA pellet was resuspended in 100 ul Ligation Buffer(66 mM Tris, pH 7.6, 6.6 mM MgCl₂, 10 mM DTT, 0.4 m ATP) plus 2 units T4DNA Ligase (Boehringer Mannheim, Indianapolis, Ind.) and then incubated18 hours at 15° C. The ligation mixture was used to transform frozencompetent KK2186 cells prepared as described by Messing, supra. Aliquotsof the ligation mixture were combined with 100 ul thawed competent cellsand held on ice for 30 minutes. The transformation mixture was heatedfor 5 minutes at 37° C. and then 0.5 ml LB containing 100 ug/mlampicillin was added.

The transformed cells were incubated 2 hours at 37° C. to allowexpression of the antibiotic resistance marker and then plated onto m9agar plates containing ampicillin, X-Gal and IPTG. Plates were incubated20 hours at 37° C. Theoretically, any plasmid carrying a DNA fragmentinserted into the Bam HI site should produce a white colony on X-Galindicator plates because this insertion interrupts the Lac Z codingsequence, however, fusions of insert sequences to Lac Z could restoreexpression of a functional B-galactosidase. For this reason, allcolonies, both white and blue, were transferred to individual wells ofmicrotiter plates containing 200 ul LB broth plus 20 ul DMSO. Thesestocks were stored at -80° C.

4. Antibody Screening of Plasmid Library Transformants.

E. coli colonies were probed in situ (Helfman, et al. (1983), Proc.Natl. Acad. Sci., U.S.A., 80:31) with bovine sera to detect expressionof cloned Pasteurella antigen genes. An eight by six pronged replicatorwas used to transfer putative transformants to a nitrocellulose diskoverlaid on an LB agar plate containing ampicillin. Plates were invertedand incubated 18 hours at 37° C. The filters, carrying bacterialcolonies, were removed and placed in a covered glass dish filled withchloroformsaturated paper toweling and held for 15 to 20 minutes to lysethe colonies. Each filter was air-dried, placed in a clean dish andincubated 18 hours with 10 ml 1× TBS, 2% milk, 1 ug DNAse, 40 uglysozyme, at room temperature. The filters were washed twice with 10 mlTBS, then incubated 2 hours at 37° C. with 200 ul bovine serum in 200 ml1× TBS, 2% milk. The filters were then washed and treated exactly asdescribed for immunodetection of proteins, as described above. Coloniesproducing immunoreactive products were purified, grown in liquidculture, and used to prepare whole cell lysates for western blotting andprotein identification, also as described above.

The predominant antigen found to be expressed by various of the plasmidtransformation exhibited a molecular weight of about 66 kilodaltons uponimmunoblot analysis. FIGS. 4A and 4B present an immunoblot of one suchclone, designated pSH200. In particular, FIGS. 4A and 4B are is animmunoblot of E. coli and P. haemolytica whole cell lysates and a P.haemolytica supernatant preparation probed with: FIG. 4A, a 1/1000dilution of convalescent bovine serum, or FIG. 4B, antigen eluted fromE. coli cells carrying pSH200 as described herein. Lanes on each gel areas follows--lane a, E. coli KK2186 (pUC7) whole cell lysate; lane b, E.coli KK2186 (PSH200) whole cell lysate; lane c, PHL101 whole celllysate; lane d, PHL101 cell-free supernatant; lane m, molecular weightmarkers.

As can be seen from FIGS. 4A and 4B, a 66 kilodalton protein, reactivewith antisera, was identified in recombinant cell pSH200 (lane b), butnot in the non-recombinant E. coli host and vector (lane a). Antibodieswith specificity for the 66K protein recognized a 66K and a slightlylarger intracellular P. haemolytica antigen and also reacted to a lesserextent with the 105K protein found in the cell-free supernatant.

E. coli KK2186 bearing recombinant plasmid, pSH200, has been depositedwith the ATCC on Nov. 25, 1987, and accorded ATCC accession number67274.

5. Construction of a P. haemolytica Library Using Bacteriophage Lambda.

Bam HI fragments of the PHL101 chromosome were used to construct alibrary using the lambda cloning vector, EMBL4 (Frischauf et al., supra(1983). EMBL4 DNA was purchased from Promega Biotec, Madison, Wis. and10 ug of this DNA was digested with 10 units Bam HI in a total volume of25 ul 20 mM Tris, pH 8.0, 100 mM NaCl, 7 mM MgCl₂, for 2 hours at 37° C.The reaction was heated for 10 minutes at 65° C. and then 1 ul 5M NaCl,2 ul 5 mM Na2EDTA, 20 ul water and 1.5 units Sal I (BRL, Bethesda, Md.)were added. The Sal I digest was incubated two hours at 37° C.; thissecond restriction digestion cleaves the non-essential "stuffer" regionof EMBL4 and is used to reduce the probability of parental bacteriophagereconstruction. Five ug of Bam HI/Sal I digested EMBL4 DNA was combinedwith 5 ug Bam HI digested P. haemolytica chromosomal DNA in a totalvolume of 100 ul. This mixture was extracted once with a 1:1 mixture ofphenol:chloroform, then once with pure chloroform, then three times withanhydrous ethyl ether. The volume of the aqueous phase was brought to150 ul and 22 ul 3M NaAc and 90 ul isopropanol were added to selectivelyprecipitate the larger DNA fragments. The precipitate was held on icefor 15 minutes, collected by centrifugation 15 minutes in an Eppendorfcentrifuge and the pellet washed once with a 1:2.5 mixture of 0.35MNaAc:ethanol. The dried pellet was resuspended in 20 ul Ligation Buffercontaining 2 units ligase, and the mixture was incubated 18 hours at 15°C.

Half of the ligation mixture was packaged, in vitro, into lambdaparticles using the Packagene Lambda DNA Packaging System purchased fromPromega Biotec, Madison, Wis. (Maniatis, et al., supra): DNA was mixedwith an entire, thawed extract, mixed gently, and then held at roomtemperature for 2 hours. 0.5 ml Phage Buffer (0.1M NaCl. 0.01M Tris, pH7.9, 0.01M MgSO₄) was added, then 25 ul chloroform, and the reaction wasvortexed to mix. The phage titer of the packaging reaction wasdetermined by plating the phage on NM538 (permissive host) and NM539(restrictive host where only recombinant phage missing the stufferfragment can form plagues), as follows. Overnight cultures of platingbacteria were harvested by centrifugation at 8000×g for 10 minutes andthe pellets resuspended in a 0.4× volume of 10 mM MgSO₄. An aliquot ordilution of the packaging extract was combined with 100 ul platingbacteria and held 20 minutes at room temperature. Phage and cells weremixed with 2.5 ml soft agar and plated directly onto LB or Lambda Agarplates and then incubated 18 hours at 37° C.

Plates having 100 or more plaques on the restrictive host, NM539, werescraped, to remove the overlay, into a Teflon centrifuge tube and theagar resuspended in 10 ml phage buffer. A 0.1 volume of chloroform wasadded and the mixture was vortexed to disperse the phage. The mixturewas held 30 minutes at 4° C. and then centrifuged 10 minutes at 1900×g.The supernatant, containing amplified recombinant phage, was removed andstored at 4° C.

6. Antibody Screening of Bacteriophage Library.

Recombinant phage producing Pasteurella proteins that could berecognized by bovine sera were detected by a direct application of thetechniques described above for immunodetection of proteins in plasmidlibrary screening. Approximately 10⁴ recombinant phage from an amplifiedstock were plated with 1.0 ml NM539, as described above, onto a 150 mmPetri plate of Lambda agar, using 10 ml 0.7% agarose instead of softagar for the overlay. The plate was incubated 1.5 hours at 37° C., toallow the lawn to develop, and then overlaid with a 137 mm 0.45 umnitrocellulose disk. Incubation was continued for 15 hours at 37° C.,after which the nitrocellulose disk was removed and incubated for 60minutes in 100 ml 1× TBS, 2% milk at 37° C. Duplicate filters wereobtained by overlaying the plate with a fresh nitrocellulose filter andincubating 10 minutes more at 37° C. After the incubation with TBS andmilk, the plaque lifts were treated exactly as described for theremaining immunodetection steps.

Plaques that gave positive responses in the primary antibody screen wereplugged from the original agar plate with a sterile Pasteur pipette into2 ml phage buffer containing 25 ul chloroform and vortexed. Theresulting solution was serially diluted in phage buffer and spotted ontofresh overlays of NM539. The resulting plaques were retested by theplaque lift and antibody screening techniques and true positives wereidentified. When necessary, these isolates were further amplified bymixing 1-4 plaques with 50 ul NM538 in a 16×150 mm culture tube, holding5 minutes at room temperature, and then adding 2 ml pre-warmed LB brothcontaining 10 mM MgSO₄. The tubes were rotated on a roller drum at 37°C. for 6 to 8 hours until lysis occurred. 0.1 ml chloroform was added toeach and the titer of each 2 ml stock was determined, using NM538 asplating bacteria (Silhavy, et al., 1984).

Immunoreactive proteins encoded by recombinant bacteriophage werefurther characterized by SDS/polyacrylamide gel electrophoresis.Bacteriophage lysates with titers as low as 10⁴ plaque forming units/mlwere used as in vivo whole cell lysates for immunological testing. Fortyul of lysate was mixed with 20 ul 3× SDS Loading Buffer and boiled 5minutes before being loaded onto and rum on a 7.5% gel, exactly asdescribed for whole cell lysates. This gel was blotted and treated withantibody and HRP-streptavidin, as previously described, and allowed adirect measurement of the size of cloned proteins.

Using the foregoing techniques, the predominant antigenic protein foundto be expressed by phage-infected recombinant cells, is a protein whichexhibits a molecular weight of approximately 55 kilodaltons byimmunoblot of a representative clone, designated clone Lambda SH-20.(shown in FIG. 5). The immunoblot was probed with convalescent serum. Inaddition to the 55K species, 5 or more smaller antigens were also seenon the immunoblot of Lambda SH20 phage lysate. These bands are notpresent in the vector control lane which implies that these antigens areencoded by the cloned DNA fragment. The smaller species are believed tobe specific degradation products of the 55K protein. A representativesample of Lambda SH-20 phage have been deposited with the ATCC on Nov.25, 1987, and accorded accession number 40285. Additionally, arepresentative sample of phage from two phage P. haemolytica clone bankshave been deposited with the ATCC. One phage clone bank, Lambda EMBL4:PhBam, constructed using total Bam HI digestion of P. haemolytica DNA,was deposited on Nov. 25, 1987, and accorded accession number 40286. Asecond phage clone bank, Lambda EMBL4: PhSau, was constructed usingpartial Sau 3A digested P. haemolytica DNA, deposited with the ATCC, andaccorded accession number 40287.

7. Purification of Cloned Pasteurella Proteins from Escherichia coliCells.

E. coli cells carrying a gene encoding a Pasteurella protein are grownto mid-logarithmic phase in LB broth or other suitable media. If thegene is controlled by the lactose promoter, isopropylthiogalactopyranoside (IPTG) is included during the logarithmic growthphase to induce transcription of the cloned gene. The cells areharvested, resuspended in 10 mM Tris, pH 7.5 and then mechanically lysedby sonication, freezing and thawing, or passage through a Frenchpressure cell. Cell debris is removed by centrifugation, 10 minutes at8000×g, and the protein-containing supernatant is concentrated byammonium sulfate or polyethylene glycol precipitation. The Pasteurellaprotein can then be purified from the concentrate by a combination ofchromatography methods.

EXAMPLE III Construction and Identification of Recombinant CellsProducing P. Haemolytica Supernatant Antigens

The present example is directed to the disclosure of an alternative andimproved method for the isolation of P. haemolytica supernatant antigensshown above in Example I and, in particular, the 105 Kilodalton antigendesignated therein as supernatant Antigen I. The method disclosed by thepresent example employs recombinant DNA techniques to clone P.haemolytica genes which encode supernatant antigens. Although thepresent example is disclosed in terms of antigen-expressing recombinantclones which are isolated from a Bgl II--P. haemolytica clone bank,there is no reason why the Sau 3A bank disclosed above would not workequally as well.

1. Preparation of P. haemolytica Restriction Fragments for Constructionof a Genomic Library.

Chromosomal DNA was prepared from P. haemolytica strain PHL101 by thelysozyme-triton lysis method described by Davis, et al., supra. A 100 mllate stationary phase culture of PHL101, grown in BHI, was harvested bycentrifugation 10 minutes at 12,000×g. The pellet was resuspended in 2ml 15% sucrose, 50 mM Tris, pH 8.5, 50 mM Na₂ EDTA, containing 1 mg/mlfresh lysozyme. The cells were incubated 60 minutes at room temperature,then 2 ml 0.1% Triton X-100 (Sigma, St. Louis, Md.), 50 mM Tris, pH 8.5,50 mM Na₂ EDTA were added. The lysate was incubated 30 minutes more atroom temperature, then 40 ul 10 mg/ml RNAse A were added and theincubation was continued for 45 minutes at 37° C.

The resulting crude lysate was used to form a six ml ethidiumbromide-CsCl density gradient (6 ml lysate, 6 g CsCl, 0.6 ml 10 mg/mlethidium bromide) (Clewell and Helinski, supra). The gradient wascentrifuged 18 hours at 60,000 rpm in a 70.1 Ti rotor. The chromosomalDNA band was located using a long wave ultraviolet light source and wasremoved from the gradient with a needle and syringe. This DNA was thensubjected to a second cycle of centrifugation through a fresh gradientand the chromosomal fraction reisolated. The ethidium bromide wasremoved by extraction with an equal volume of isopropanol and theresulting DNA solution was dialyzed 16 hours, 4° C. versus 100 volumes1× TE Buffer (10 mM Tris, pH 8.0, 1 mM Na₂ EDTA).

For library construction, the PHL101 chromosomal DNA was digestedcompletely with Bgl II, as follows. 20 ug PHL101 chromosomal DNA weredigested with 40 units Bgl II (BRL, Bethesda, Md.), in 100 ul 20 mMTris, pH 7.6, 50 mM NaCl, 7 mM MgCl₂, for 2 hours at 37° C. The reactionwas terminated by heating 10 minutes at 65° C. and the digest was storedat -20° C.

2. Construction of a P. Haemolytica Library Using Bacteriophage Lambda.

Bgl II fragments of the PHL101 chromosome were used to construct alibrary using the lambda cloning vector, EMBL4 (Frischauf, et al.,supra). EMBL4 DNA was purchased from Promega Biotec, Madison, Wis. and10 ug of this DNA was digested with 10 units Bam HI in a total volume of25 ul 20 mM Tris, pH 8.0, 100 mM NaCl, 7 mM MgCl₂, for 2 hours at 37° C.The reaction was heated for 10 minutes at 65° C. and then 1 ul 5M NaCl,2 ul 5 mM Na₂ EDTA, 20 ul water and 1.5 units Sal I (BRL, Bethesda, Md.)were added. The Sal I digest was incubated two hours at 37° C.; thissecond restriction digestion cleaves the non-essential "stuffer" regionof EMBL4 and was used to reduce the probability of parentalbacteriophage reconstruction. Five ug of Bam HI/Sal I digested EMBLA DNAwas combined with 5 ug Bgl II-digested P. haemolytica chromosomal DNa ina total volume of 100 ul.

This mixture was extracted once with a 1:1 mixture of phenol:chloroform,then once with pure chloroform, then three times with anhydrous ethylether. The volume of the aqueous phase was brought to 150 ul and 22 ul3M NaAc and 90 ul isopropanol were added to selectively precipitate thelarger DNA fragments. The precipitate was held on ice for 15 minutes,collected by centrifugation 15 minutes in an Eppendorf centrifuge andthe pellet washed once with a 1:2.5 mixture of 0.35M NaAc:ethanol. Thedried pellet was resuspended in 20 ul Ligation Buffer containing 2 unitsligase, and the mixture was incubated 18 hours at 15° C.

Half of the ligation mixture was packaged, in vitro, into lambdaparticles using the Packagene Lambda DNA Packaging System purchased fromPromega Biotech, Madison, Wis. (Maniatis, et al., supra): DNA was mixedwith an entire thawed extract, mixed gently, and then held at roomtemperature for 2 hours. 0.5 ml Phage Buffer (0.1M NaCl, 0.01M Tris, pH7.9, 0.01M MgSO₄) was added, then 25 ul chloroform, and the reaction wasvortexed to mix. The phage titer of the packaging reaction wasdetermined by plating the phage on NM538 (permissive host) and NM539(restrictive host where only recombinant phage missing the stufferfragment can form plaques), as follows.

Overnight cultures of plating bacteria were harvested by centrifugationat 8000×g for 10 minutes and the pellets resuspended in a 0.4× volume of10 mm MgSO₄. An aliquot or dilution of the packaging extract wascombined with 100 ul plating bacteria and held 20 minutes at roomtemperature. Phage and cells were mixed with 2.5 ml soft agar and plateddirectly onto LB or Lambda Agar plates and then incubated 18 hours at37° C. Plates having 100 or more plaques on the restrictive host, NM539,were scraped, to remove the overlay, into a teflon centrifuge tube andthe agar resuspended in 10 ml Lambda Diluent (10 mM Tris, pH 7.6, 10 mMMgSO₄, 1 mM Na₂ EDTA. A 0.1 volume of chloroform was added and themixture was vortexed to disperse the phage. The mixture was held 30minutes at 4° C. and then centrifuged 10 minutes at 1900×g. Thesupernatant, containing amplified recombinant phage, was removed andstored at 4° C.

3. Antibody Screening of Bacteriophage Librarv.

Recombinant phage producing Pasteurella proteins that could berecognized by bovine sera were detected by a direct application of thetechniques described above for immunodetection of proteins and plasmidlibrary screening. Approximately 10⁴ recombinant phage from an amplifiedstock were plated with 1.0 ml NM539, as described above, onto a 150 mmPetri plate of Lambda agar, using 10 ml 0.7% agarose instead of softagar for the overlay. The plate was incubated 1.5 hours at 37° C., toallow the lawn to develop, and then overlaid with a 137 mm 0.45 umnitrocellulose disk. Incubation was continued for 15 hours at 37° C.,after which the nitrocellulose disk was removed and incubated for 60minutes in 100 ml 1× TBS, 2% milk at 37° C. Duplicate filters wereobtained by overlaying the plate with a fresh nitrocellulose filter andincubating 10 minutes more at 37° C. After the incubation with TBS andmilk, the plaque lifts were treated exactly as described for theremaining immunodetection steps.

Plaques that gave positive responses in the primary antibody screen wereplugged from the agar with a sterile Pasteur pipette into 2 ml LambdaDiluent containing 25 ul chloroform and vortexed. The resulting solutionwas serially diluted in Lambda Diluent and spotted onto fresh overlaysof NM539. These plaques were retested by the plaque lift and antibodyscreening techniques and true positives were identified. When necessary,these isolates were further amplified by mixing 1-4 plaques with 50 ulNM538 in a 16×150 mm culture tube, holding 5 minutes at roomtemperature, and then adding 2 ml pre-warmed 1B broth containing 10 mMMgSO₄. The tubes were rotated on a roller drum at 37° C. for 6 to 8hours until lysis occurred. 0.1 ml chloroform was added to each and thetiter of each 2 ml stock was determined, using NM538 as platingbacteria.

Immunoreactive proteins encoded by recombinant bacteriophage werefurther characterized by SDS/polyacrylamide gel electrophoresis.Bacteriophage lysates with titers as low as 10⁴ plaque forming units/mlwere used as in vivo whole cell lysates for immunological testing. Fortyul of lysate was mixed with 20 ul 3× SDS Loading Buffer and boiled 5minutes before being loaded onto and run on a 7.5% gel, exactly asdescribed for whole cell lysates. This gel was blotted and treated withantibody and HRP-streptavidin, as previously described, and allowed adirect measurement of the size of cloned proteins.

Antibody screening of the Bql II recombinant phage library provided 34positive single-plague isolates. Following purification andamplification, crude phage lysates of the isolates were tested forantigen production by Western blotting using bovine immune serum. Eightof the 34 original isolates produced an antigen that was visible on animmunoblot. Seven of these produced the same 66 kD antigen that waspreviously identified as being encoded by plasmid pSH200 from the pUC7plasmid library. Southern blot analysis verified that these sevenisolates carried the same 3.0 kb Eco RI fragment carried by pSH200. Theremaining recombinant phage, Lambda SH132, produced an antigen having anapparent molecular weight of 105 kD. (See FIG. 6) This antigencorresponds to Antigen Group I of the P. haemolytica supernatant antigengroups. Antibodies eluted from immunoblots of Lambda SH132 were able torecognize Antigen I, indicating that these proteins were antigenicallyidentical.

4. Subcloning and Mapping the 105 kD Antigen Gene.

To facilitate mapping and expression of the 105 kD antigen gene, the P.haemolytica DNA insert from lambda SH132 was cloned into the plasmidvector, pBS (+) (Stratagene, San Diego, Calif.). Restriction analysis ofLambda SH132 indicated that the insert was cut once by Eco RI, yielding1.2 and 17.6 kb fragments. Therefore, Eco RI was used to cut LambdaSH132 for subcloning, as follows.

One ug of Lambda SH132 was digested with 10 units Eco RI 2 hours at 37°C. in a 50 ul reaction containing 100 mm Tris, pH 7.5, 10 mM MgCl₂, 50mM NaCl. Five ug pBS (+) were similarly digested. To prevent religationof the vector, the digested pBS (+) DNA was treated with calf intestinalphosphatase.(CIP). The digested Lambda SH132 DNA was combined with oneug Eco RI-linearized pBS (+). The DNAs were coprecipitated and thenligated, as previously described.

One-half of the ligation mixture was used to transform competent KK2186cells with selection at 30° C. on LB plates containing ampicillin andX-Gal. Cells carrying plasmids with inserts were identified as whitecolonies on these indicator plates. Restriction digest analysis ofplasmid DNAs prepared from these isolates indicated that three differentplasmid constructs had been generated: pSH207 (pBS::Lambda SH132 17.6 kbEco RI, orientation A), pSH209 (pBS::Lambda SH132 17.6 kb Eco RI,orientation B), and pSH210 (pBS:: Lambda SH132 1.2 kb Eco RI).Restriction maps for these three constructs are shown in FIG. 7A.

Whole cell lysates of strains carrying the plasmids were prepared andtested by Western blotting to show that the 17.6 kb Eco RI fragmentproduced the 105 kD antigen (FIG. 7B). Plasmid pSH210 did not produce anantigen while pSH209 produced more of the 105 kD antigen than didpSH207. This suggested that the expression of the 105 kD antigen genecould be influenced by vector sequences that flanked the insert, e.g. bythe Lac promoter on pBS (See following section of Antigen Production).

The location of the gene was further mapped within the 17.6 kb Eco RIfragment by constructing in vitro deletions of pSH207 and then testingdeletants for antigen production by Western blotting. Deletions wereconstructed by digesting 1-5 ug pSH207 with either Ava I, Hinc II, PstI, or double digests of Bam HI plus Bgl II or Acc I plus Cla I understandard digestion conditions. The digested DNAs were phenol extracted,ethanol precipitated and then resuspended in Ligation Buffer and ligatedwith T4 DNA ligase, as previously described.

The ligated DNAs were transformed into competent KK2186 cells withselection for ampicillin resistance. Plasmid DNA was prepared fromtransformants corresponding to each deletion type and screened for theloss of the expected DNA fragments by restriction digest analysis. Mapsof the resulting deletants are shown in FIG. 7A. The deletant plasmidsare: pSH214 (Hinc II deletion), pSH215 (Pst I deletion), pSH216 (1.2 kbAva I deletion), pSH217 (Acc I--Cla I deletion), pSH218 (Bam HI--Bgl IIdeletion) and pSH219 (1.2, 1.4 kb Ava I deletion). Whole cell extractsof the strains carrying the deletant plasmids were tested for theproduction of the 105 kD antigen by Western blot analysis, as before(FIG. 7B).

The nucleotide sequence of the ptx gene was determined using singlestranded templates from subclones in M13 and pBS vectors, and the T7 DNApolymerase, Sequenase kit of United States Biochemicals (Cleveland,Ohio). The nucleotide sequence determined was subjected to computersequence analysis using the Pustell DNA Sequence Analysis Program ofInternational Biotechnologies, Inc. (New Haven, Conn.), from which wasdeduced the amino acid sequences encoded by the DNA that was analyzed.

Shown in FIGS. 9A-9L is a DNA sequence extending from a BamHI/BglIIcloning junction including the sequence determined for the leukotoxin(105K antigen) gene and additional sequences represented within theoriginal lambda SH132 clone, as well as in plasmid pSH209.

5. Antigen Production and Leukotoxin Activity.

Since it was observed that the level of expression of the 105 kD antigenwas orientation dependent, it was anticipated that the Lac promoter onthe Bluescribe vector was contributing to the transcription of thecloned Pasteurella gene. Strain KK2186 produces Lac repressor andmaintains a low level of Lac promoted transcription; in the presence ofIPTG, transcription is induced. Therefore, cells carrying either pSH207or pSH209 were grown in the presence and in the absence of IPTG todetermine if the expression of the 105 kD Antigen I could be induced.Cells were grown at 30° C. in LB broth containing ampicillin to aturbidity of 150 Klett units. The cultures were split and IPTG was addedto one half of each culture to 0.5 mM. Growth at 30° C. was continuedfor 3 hours and then the cells were harvested by centrifugation. Wholecell extracts were prepared and subjected to Western blot analysis, asbefore. FIG. 8 shows that production of the antigen was increased atleast 10-fold by IPTG from pSH209, while IPTG did not significantlyincrease expression from pSH207. This enhanced expression under controlof an inducible promoter has allowed us to produce relatively largequantities of the 105 kD antigen for other studies.

Whole cell extracts of IPTG-induced E. coli cells carrying pSH209 wereused in an assay to demonstrate that the 105 kD antigen possessedmacrophage killing (leukotoxic) activity, as follows. Ten mls freshbovine blood were diluted to 25 mls in 1× Hanks Balanced Salt Solution(HBSS), then 25 mls Ficoll-Hypaque were layered under the bloodsolution. This was spun 40 minutes at approximately 1500×g, at roomtemperature, to form a gradient. The top layer of the gradient wasdiscarded and the second layer of the gradient, containing lymphocytes,was removed and saved. Similarly, the third layer was discarded, whilethe fourth layer, containing neutrophils was removed and saved. Ammoniumchloride was added to the neutrophil and lymphocyte fractions to 0.43%and the mixtures were incubated 5 min. at room temperature to lyse anycontaminating red blood cells. Each fraction was diluted to 50 ml inRPMI 1640 medium and the cells pelleted by centrifugation 40 minutes at1500×g. This washing step was repeated twice more. Final cell volumeswere approximately 10 ml, with each fraction containing about 10⁷cells/ml.

To measure toxic activity, 2×10⁶ neutrophils were mixed with variousdilutions of sonicated whole cell lysates (10 minutes, 20 watts) ofIPTG-induced E. coli strains carrying either pBS + or pSH209 Cells plussonicates were incubated 30 minutes at 37° C. then stained with Trypanblue and viable cells counted using light microscopy. As shown in TableII below, whereas an undiluted sonicate of the pBS + carrying strain didnot cause any loss of neutrophil viability, the sonicate of the pSH209carrying strain (SH027) killed 65% of these cells. As expected, neithersonicate caused any killing of lymphocytes.

                  TABLE II    ______________________________________    Cytotoxic Activity of Pasteurella Toxin    Produced in P. haemolytica and E. coli.                      % Neutrophil Death    ______________________________________    P. haemolytica supernatant                        60.4    P. haemolytica whole cell sonicate                        61.7    E. coli (PSH209) whole cell sonicate                        64.7    (IPTG induced culture)    E coli (pBS) whole cell sonicate                         0.1    (IPTG induced culture)    Control (medium only)                         0.0    ______________________________________

6. Large Scale Preparation of Membrane-Associated Leukotoxin and VaccineCompositions.

Strain SH027 carrying pSH209 was grown and IPTG-induced, as describedabove, in 250 ml L broth. Cells were collected by centrifugation, 5 min.at 10,000 rpm, and resuspended to a density of 7×10⁹ cells/ml in 0.75Msucrose, 10 mM Tris, pH 7.8 containing 100 ug/ml lysozyme. This solutionwas incubated 2 min. on ice then slowly diluted with two volumes 1.5 mMEDTA, pH 7.5 over a period of 10 minutes. The spheroplasts formed bythis procedure were osmotically shocked by pouring the suspension into 4volumes ice cold water and stirring for 10 minutes at 4° C. Unbrokencells were removed from other cellular components by centrifugation at1200×g, 15 minutes at 4° C. Membranes were collected by centrifuging thesupernatant fraction at 60,000 rpm for 2 hours at 4° C. in a 70 Ti fixedangle rotor.

The membrane pellet was resuspended in 40 ml 0.25M sucrose, 33 mM Tris,pH 7.8, 1 mM EDTA and repelleted 2 hours at 60,000 rpm. The washedpellet was resuspended in 2.0 ml 25% sucrose, 5mM EDTA, pH 7.5 andoverlayed onto a gradient composed of steps with the following sucroseconcentrations and volumes: 55%, 5.0 ml; 50%, 6.3 ml; 45%, 6.3 ml; 40%,6.3 ml; 35%, 6.3 ml; 30%, 6.3 ml; 25%, 6.3 ml. Gradients werecentrifuged 24 hours at 35,000 rpm in a SW 41 rotor at 4° C. and thenfractioned into 0.8 ml; fractions. Each fraction was diluted with 1 mMEDTA, pH 7.5, to a sucrose concentration of less than 10% and thenconcentrated by pelleting 2 hours at 65,000 rpm, 4° C. in a 70.1 Tirotor. The pellets were resuspended in minimal volumes of 33 M Tris, pH7.8 and the entire sample of each was used for Western blot analysis.This analysis indicated that the 105 kD leukotoxin was associated withboth the inner and outer membranes of E. coli.

Vaccine compositions may include extracts of strain SH027 taken atseveral stages of the above purification scheme. For example, asonicated whole cell extract of IPTG-induced cells, a crude membranepellet, or purified inner and/or outer membranes may be used inconjunction with a suitable pharmaceutical carrier. In addition, avaccine may be composed of any of the above, alone or in combination,mixed with any or all of the crude or cloned P. haemolytica supernatantantigens; this includes the 55 kD and 66 kD antigens that have also beencloned in E. coli.

D. Vaccine Preparation

Immunogenic compositions, suitable for use as a Shipping Fever vaccine,may be prepared most readily directly from P. haemolytica cell-freeculture supernatant, by, for example, ammonium sulfate precipitation ofsupernatant proteins, to concentrate the proteins, followed by extensivedialysis to remove undesired small molecular weight molecules and/orlyophilization of the thus purified material for more ready formulationinto a desired vehicle. There is no general requirement that thesupernatant be molecular weight fractionated to provide the individualantigens in their most purified state because it has been found that theunfractionated supernatant itself will provide the antigens in asufficiently substantially purified form to elicit an immune response inanimals receiving such an immunogenic composition.

Alternatively, one may desire to formulate immunogen compositions usingthe antigen complex derived by gel exclusion chromatography of thesupernatant proteins, which complex represents a more substantiallypurified antigen preparation relative to the supernatant. Alternatively,antigens derived by antibody-Sepharose chromatography of supernatantantigens may be employed. In an even more preferred embodiment, one ormore, but preferably all, of the individual, isolated antigens areemployed to prepare an antigenic protein "cocktail". Such cocktails maybe prepared by the admixture of approximately equimolar amounts of thepurified proteins, or alternatively, through the admixture ofequi-antigenic amounts of one or more of the antigens.

In still further embodiments, immunogen compositions may be formulatedto include one or both of the antigens produced by the recombinant cellsof the present invention, these antigens being included in optimalamounts, for example, approximately equimolar or equi-antigenic amounts.

The preparation of vaccines which contain peptide sequences as activeingredients is generally well understood in the art, as exemplified byU.S. Pat. Nos. 4,608,251; 4,601,903; 4,599,231; 4,599,230; 4,596,792;and 4,578,770, all incorporated herein by reference. Typically, suchvaccines are prepared as injectables. Either as liquid solutions orsuspensions: solid forms suitable for solution in, or suspension in,liquid prior to injection may also be prepared. The preparation may alsobe emulsified. The active immunogenic ingredient is often mixed withexcipients which are pharmaceutically acceptable and compatible with theactive ingredient. Suitable excipients are, for example, water, saline,dextrose, glycerol, ethanol, or the like and combinations thereof. Inaddition, if desired, the vaccine may contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents. pH buffering agents,or adjuvants which enhance the effectiveness of the vaccine.

The vaccines are conventionally administered parenterally, by injection,for example, either subcutaneously or intramuscularly. Additionalformulations which are suitable for other modes of administrationinclude suppositories and, in some cases, oral formulations. Forsuppositories, traditional binders and carriers may include, forexample, polyalkalene glycols or triglycerides: such suppositories maybe formed from mixtures containing the active ingredient in the range of0.5% to 10%, preferably 1-2%. Oral formulations include such normallyemployed excipients as, for example, pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharine, cellulose,magnesium carbonate and the like. These compositions take the form ofsolutions, suspensions, tablets, pills, capsules, sustained releaseformulations or powders and contain 10-95% of active ingredient,preferably 25-70%.

The proteins may be formulated into the vaccine as neutral or saltforms. Pharmaceutically acceptable salts, include the acid additionsalts (formed with the free amino groups of the peptide) and which areformed with inorganic acids such as, for example, hydrochloric orphosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups mayalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine. 2-ethylamino ethanol,histidine, procaine, and the like.

The vaccines are administered in a manner compatible with the dosageformulation, and in such amount as will be therapeutically effective andimmunogenic. The quantity to be administered depends on the subject tobe treated, capacity of the cow's immune system to synthesizeantibodies, and the degree of protection desired. Precise amounts ofactive ingredient required to be administered depend on the judgment ofthe practitioner and are peculiar to each individual cow. However,suitable dosage ranges are of the order of several hundred microgramsactive ingredient per animal. Suitable regimes for initialadministration and booster shots are also variable, but are typified byan initial administration followed by subsequent inoculations or otheradministrations.

The manner of application may be varied widely. Any of the conventionalmethods for administration of a dead vaccine are applicable. Theseinclude oral application on a solid physiologically acceptable base orin a physiologically acceptable dispersion, parenterally, by injectionor the like. The dosage of the vaccine will depend on the route ofadministration and will vary according to the size of the host. Becausethe vaccine is believed to have few if any side effects, relativelylarge dosages may be used without injury to the cow. Normally, theamount of the vaccine will be form about 1 mu g to 20.0 mg per kilogramof host, more usually from about 5 mu g to 2.0 mg given subcutaneouslyor intramuscularly after mixing with an appropriate carrier or anadjuvant to enhance immunization with the vaccine.

Various methods of achieving adjuvant effect for the vaccine includesuse of agents such as aluminum hydroxide or phosphate (alum), commonlyused as 0.05 to 0.1 percent solution in phosphate buffered saline,admixture with synthetic polymers of sugars (Carbopol) used as 0.25percent solution, aggregation of the protein in the vaccine by heattreatment with temperatures ranging between 70° to 101° C. for 30 secondto 2 minute periods respectively. Aggregation by reactivating withpepsin treated (Fab) antibodies to albumin, mixture with bacterial cellssuch as C. parvum or endotoxins or lipopolysaccharide components ofgram-negative bacteria, emulsion in physiologically acceptable oilvehicles such as mannide mono-oleate (Aracel A) or emulsion with 20percent solution of a perfluorocarbon (Fluosol-DA) used as a blocksubstitute may also be employed.

More novel methods of adjuvanticity would include attenuated bacterialtoxins against which the host has been preimmunized, or, by including inthe vaccine composition a biologically or antigenically sufficientamount of P. haemolytica bacterin. Bacterin preparation is well known inthe art and basically involves formalinization of live P. haemolyticacells as follows. Briefly, P. haemolytica cultures are grown in BrainHeart Infusion broth to mid-logarithmic phase then harvested andresuspended in phosphate buffered saline (PBS) to an equivalent celldensity. The cells are then incubated overnight at room temperature inthe presence of 0.5% formalin, reharvested and resuspended in PBS.Aliquots of the formalinized bacteria are mixed with aluminum hydroxide,incomplete Preund's or other suitable adjuvants and then used toinoculate test animals.

For best results it is believed that a weight ratio of about 1:1 to 1:5,bacterin:supernatant (or isolated supernatant proteins), respectively,should be employed.

The compound vaccine should elicit enhanced immune response to P.haemolytica infection. The amount of the adjuvant will vary widelydepending upon the nature of the adjuvant, generally varying from 0.1 to100 times the weight of the immunogen, more usually from 1 to 10 times.

In many instances, it will be desirable to have multiple administrationsof the vaccine, usually not exceeding six vaccinations, more usually notexceeding four vaccinations and preferably one or more, usually at leastabout three vaccinations. The vaccinations will normally be at from twoto twelve week intervals, more usually from three to five weekintervals. Periodic boosters at intervals of 1-5 years, usually threeyears, will be desirable to maintain protective levels of theantibodies. The course of the immunization may be followed by assays forantibodies for the supernatant antigens. The assays may be performed bylabelling with conventional labels, such as radionuclides, enzymes,fluorescers, and the like. These techniques are well known and may befound in a wide variety of patents, such as U.S. Pat. Nos. 3,791,932;4,174,384 and 3,949,064, as illustrative of these types of assays.

E. Antibody Generation and Further Embodiments

Antibodies to one or more of the P. haemolytica antigens, orantigen-containing compositions, may be obtained, in general, throughimmunization of a selected immuno-competent mammal with the antigen orcomposition, as the case may be. Satisfactory immunization protocols arewell known, and have been dealt with extensively herein. However, itshould be pointed out that the antibodies elicited in response toinoculation with P. haemolytica antigens have additional utility in andof themselves. For example, either polyclonal or monoclonal antibodies,regardless of species. derivation, can be employed in ELISA, or similarimmunodetection assays, for the diagnosis of active or convalescentPasteurellosis.

Additionally, immunized bovine sera can be fractionated to providehighly Pasteurella-immunoreactive sera in the form of bovinegamma-globulins.

To provide a general purpose anti-Pasteurella antibody composition,immunocompetent mammals are inoculated with one of the individualantigens or antigen compositions disclosed herein, typically togetherwith a suitable immuno-adjuvant, in a manner sufficient to elicit aPasteurella antigen-specific immune response. Generally, the amount ofantigen material employed will be chosen as is required under theindividual circumstances. After a satisfactory response has beenobtained, as gauged by immunoblot, ELISA or other immunologic detectiontest, an aliquot of blood is removed from the animal and the serumobtained therefrom. The serum is then fractionated, for example, byammonium sulfate precipitation and dialysis, to provide the serum Igfraction or subfractions thereof.

For more specific application, for example, for use in inducing apassive immunity to the disease, a pasteurella-specific hyperimmunebovine serum fraction is provided by inoculating a cow in a manner, forexample, as detailed herein in Example I or Section D. A satisfactoryimmune response, as gauged by one of the various immunologic tests, willtypically be obtained within 3 to 6 weeks, and may be further enhancedby repeated booster inoculations on a weekly basis. The resultanthyperimmune serum is then obtained and fractionated to provide,typically, the gamma globulin fraction. After suitable purification, forexample, further dialysis or fractionation, the immunoglobulins may beformulated into a suitable pharmaceutical vehicle for parentaladministration. Depending on the immunoglobulin concentration and titer,generally 5 to 10 cc will be administered to animals, for example, highrisk cattle exposed to the disease or being subjected to conditionswhich are conductive to the disease (high density containment, shipping,etc.) For more specific purposes, for example, for the immunodetectionof specific, P. haemolytica individual antigens, one may desire togenerate a hybridoma population which secreted monoclonal antibodieshaving specificity in general for P. haemolytica antigens, and selectingtherefrom clones having specificity in particular for the individualantigens which have been identified herein.

Hybridoma development is well known, as exemplified by theaforementioned U.S. Pat. No. 4,196,265, and involves, in general, firstimmunizing a rodent, for example, a mouse or rat, with a selectedantigen or antigen composition obtained in accordance with the presentinvention, in a manner sufficient to provide a satisfactory immuneresponse. Spleen cells from the immunized animal are then fused withmyeloma cells of the corresponding species. Typically, one may desire toemploy immunocompetent mice and murine NS-1 myeloma cells.

The fused spleen/myeloma cells are then subjected to culturing in aselective medium, for example, HAT media (hypoxanthine, aminopterin,thymidine), to select fused spleen/myeloma cells from the parentalcells. This culturing, in essence, provides the population of hybridomasfrom which specific hybridomas are selected. Typically, selection isperformed by culturing the cells by single-clone dilution intomicrotiter plates, followed by testing the individual clonalsupernatants for reactivity with one of the individual antigens. Mostconveniently, the clonal supernatants are first screened by ELISA toidentify as a whole those colonies reactive with P. haemolyticaantigens, and then individual reactive colonies are screened byimmunoblot to determine the antigenic specificity of the particularmonoclonal antibody produced by each individual colony. The selectedcolony may then be propagated indefinitely to provide the monoclonalantibody containing supernatant.

The monoclonal or polyclonal antibodies may thus be provided in a formconvenient for application in one of the conventional immunologic assay,for the detection of corresponding P. haemolytica antigens in variousfluids, for example, biologic fluids obtained from cattle.

Alternatively, antibodies may be employed for specific isolation ofindividual P. haemolytica antigens, for example, by attachment toSepharose and chromatography of antigen-containing compositionsthereover. Individual antigen-specific monoclonal antibodies may thus beemployed to isolate individual antigens for antigenic "cocktail"formulation.

It is believed that, for diagnostic application, preferredpasteurellosis diagnostic methods would employ P. haemolytica antigens,whether isolated or employed in the form of purified P. haemolyticasupernatants, to immunoidentify the presence of P. haemolyticaantibodies in biologic samples, tissue or fluids, obtained from asuspected infected animal. For detection in aqueous samples, theantigen, or antigen composition, is preferably adsorbed, or otherwiseattached, to an appropriate adsorption matrix, for example, the insidesurface of a microtiter dish well, and an aqueous suspectedantibody-containing composition contacted therewith in a mannersufficient to promote specific immunocomplex formation. The matrix isthen washed to remove non-specifically bound material and the amount ofmaterial which is specifically immunocomplexed thereto determined,typically through the use of an appropriate labeled ligand.

For the determination of an active infection, one may desire to furtherprobe specific for bovine IgM molecules. It is known that a relativeincrease in the proportion of specific IgM is often indicative of anactive, as opposed to convalescent or otherwise non-active, disease.

Accordingly, diagnostic kits may be developed which include aliquots ofone or more of the P. haemolytica antigens, or antigen-containingcompositions, which are, preferably, provided in a form which issuitable for application to microtiter dish wells. Alternatively, or inaddition, the kits will include antibody compositions having specificityfor one or more of the antigens. Both antibody and antigen preparationsshould preferably be provided in a suitably titrated form, with antigenconcentrations and/or antibody titers given for easy reference inquantitative applications.

The kits will also typically include an immunodetection reagent or labelfor the detection of specific immunoreaction between the providedantigen and/or antibody, as the case may be, and the diagnostic sample.Suitable detection reagents are well known in the art as exemplified byradioactive, enzymatic or otherwise chromogenic ligands, which aretypically employed in association with the antigen and/or antibody, orin association with a second antibody having specificity for the antigenor first antibody. Thus, the reaction is detected or quantified by meansof detecting or quantifying the label. Immunodetection reagents andprocesses suitable for application in connection with the novelcompositions of the present invention are generally well known in theart.

    __________________________________________________________________________    #             SEQUENCE LISTING    - (1) GENERAL INFORMATION:    -    (iii) NUMBER OF SEQUENCES: 2    - (2) INFORMATION FOR SEQ ID NO:1:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 10 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    - Met Gly Thr Arg Leu Thr Thr Leu Ser Asn    #                10    - (2) INFORMATION FOR SEQ ID NO:2:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 10 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    - Leu Ser Ser Leu Gln Phe Ala Arg Ala Ala    #                10    __________________________________________________________________________

What is claimed is:
 1. A process for preparing a P. haemolytica antigencomprising the steps of:(a) culturing P. haemolytica bacteria to producea cell-free supernatant without lysing the bacteria, the supernatanthaving individual P. haemolytica secreted polypeptides; (b) subjectingpolypeptides of the culture supernatant to molecular weightfractionation to fractionate the polypeptides according to theirmolecular weight; (c) identifying an antigen having binding affinity forimmune sera from pasteurellosis infected cows; and (d) purifying theidentified antigen wherein the identified antigen has a molecular weightof approximately 105 kD, as determined by SDS polyacrylamide gelelectrophoresis.
 2. A process for preparing a P. haemolytica antigencomprising the steps of:(a) obtaining an antibody preparation whichincludes antibodies against the P. haemolytica antigen; (b) preparing animmunoaffinity chromatography substrate from the antibody preparation;(c) culturing P. haemolytica bacteria to produce a cell-free culturesupernatant without lysing the bacteria, the supernatant havingindividual P. haemolytica secreted polypeptides; and (d) immunopurifyingthe antigen from the cell-free culture supernatant by immunoaffinitychromatography of the supernatant on the immunoaffinity chromatographysubstrate wherein the antigen has a molecular weight of approximately105 kD, as determined by SDS polyacrylamide gel electrophoresis.
 3. Theprocess of claim 2, wherein the antibody preparation comprises anantibody prepared in vitro against an antigen composition having thefollowing properties:(a) binding affinity for immune sera obtained froma pasteurellosis infected cow; (b) an approximate reference molecularweight of 105 K Daltons, the molecular weight being ascertainable by SDSpolyacrylamide gel electrophoresis and immunoblot analysis; and (c)immunological cross-reactivity with a 105 K Dalton P. haemolyticaantigen having an amino terminal sequence of M-G-T-R-L-T-T-L-S-N- (SEQID NO:1) and a carboxy terminal sequence of -L-S-S-L-Q-F-A-R-A-A (SEQ IDNO: 2).
 4. A process for preparing a P. haemolytica cytotoxin antigenpreparation comprising the steps of:(a) culturing P. haemolyticabacteria to produce a supernatant, the supernatant comprising individualP. haemolytica secreted proteins; and (b) subjecting said supernatant tofractionation to enrich for proteins having a molecular weight greaterthan 15 Kd, while substantially retaining the cytotoxin antigen, whereinthe cytotoxin antigen is defined as having the followingproperties:binding affinity for immune sera obtained from apasteurellosis infected cow; and an approximate reference molecularweight of 105 Kd, the molecular weight being ascertainable by SDSpolyacrylamide gel electrophoresis and immunoblot analysis.
 5. Theprocess of claim 4, further comprising concentration of the supernatant.6. The process of claim 4, further comprising subjecting the antigenpreparation to ion exchange chromatography or gel filtrationchromatography.
 7. The process of claim 4, further comprising purifyingthe antigen preparation so as to decrease the amount of protein antigenshaving a molecular weight less than about 29 Kd.
 8. The antigen of claim7, wherein the antigen preparation is purified so as to decrease theamount of protein antigens having a molecular weight less than about 73Kd.
 9. A process for preparing a purified antigen having the followingproperties:binding affinity for immune sera obtained from apasteurellosis infected cow; an approximate reference molecular weightof 105 K Daltons, the molecular weight being ascertainable by SDSpolyacrylamide gel electrophoresis and immunoblot analysis; andimmunological cross-reactivity with a 105 K Dalton P. haemolyticaantigen found in cell-free supernatant following growth of P.haemolytica cells in culture;wherein the process comprises preparing theantigen by: a) culturing P. haemolytica bacteria to produce a cell-freesupernatant, the supernatant comprising individual P. haemolyticasecreted proteins; andi) subjecting said supernatant to concentrationand dialysis using a dialysis membrane having a molecular weight cutoffof at least 15 Kd; ii) subjecting the supernatant to ammonium sulfateprecipitation, iii) subjecting the supernatant to gel exclusionchromatography; or iv) subjecting the supernatant to ion exchangechromatography; and b) recovering said antigen.
 10. The process of claim4, further comprising concentrating the supernatant or preparation bylyophilization.
 11. The process of claim 4, wherein fractionation isachieved by dialysis.
 12. The process of claim 4, wherein fractionationis achieved by filtration.
 13. The process of claim 4, furthercomprising rendering the preparation pharmaceutically acceptable.